Anti-GL50 antibodies

ABSTRACT

The invention provides isolated nucleic acids molecules, designated GL50 nucleic acid molecules, which encode novel GL50 polypeptides. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing GL50 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a GL50 gene has been introduced or disrupted. The invention still further provides isolated GL50 polypeptides, fusion proteins, antigenic peptides and anti-GL50 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/155,043, filed onSep. 21, 1999. The entire contents of that application are herebyincorporated in their entirety by this reference.

BACKGROUND OF THE INVENTION

In order for T cells to respond to foreign proteins, two signals must beprovided by antigen-presenting cells (APCs) to resting T lymphocytes(Jenkins, M. and Schwartz, R. (1987) J. Exp. Med 165:302-319; Mueller,D. L. et al. (1990) J. Immunol. 144:3701-3709). The first signal, whichconfers specificity to the immune response, is transduced via the T cellreceptor (TCR) following recognition of foreign antigenic peptidepresented in the context of the major histocompatibility complex (MHC).The second signal, termed costimulation, induces T cells to proliferateand become functional (Lenschow et al. (1996) Annu. Rev. Immunol.14:233). Costimulation is neither antigen-specific, nor MHC restrictedand is thought to be provided by one or more distinct cell surfacemolecules expressed by APCs (Jenkins, M. K. et al. (1988) J. Immunol.140:3324-3330; Linsley, P. S. et al. (1991) J. Exp. Med 173:721-730;Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6575-6579;Young, J. W. et al. (1992) J. Clin. Invest 90:229-237; Koulova, L. etal. (1991) J. Exp. Med. 173:759-762; Reiser, H. et al. (1992) Proc.Natl. Acad. Sci. USA 89:271-275; van-Seventer, G. A. et al. (1990) J.Immunol. 144:4579-4586; LaSalle, J. M. et al. (1991) J. Immunol.147:774-80; Dustin, M. I. et al. (1989) J. Exp. Med 169:503; Armitage,R. J. et al. (1992) Nature 357:80-82; Liu, Y. et al. (1992) J. Exp. Med.175:437-445).

The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs, arecritical costimulatory molecules (Freeman et al. (1991) J. Exp. Med.174:625; Freeman et al. (1989) J. Immunol. 143:2714; Azuma et al. (1993)Nature 366:76; Freeman et al. (1993) Science 262:909). B7-2 appears toplay a predominant role during primary immune responses, while B7-1,which is upregulated later in the course of an immune response, may beimportant in prolonging primary T cell responses or costimulatingsecondary T cell responses (Bluestone (1995) Immunity 2:555).

One ligand to which B7-1 and B7-2 bind, CD28, is constitutivelyexpressed on resting T cells and increases in expression afteractivation. After signaling through the T cell receptor, ligation ofCD28 and transduction of a costimulatory signal induces T cells toproliferate and secrete IL-2 (Linsley, P. S. et al. (1991) J. Exp. Med173:721-730; Gimmi, C. D. et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; June, C. H. et al. (1990) Immunol. Today 11:211-6;Harding, F. A. et al. (1992) Nature 356:607-609). A second ligand,termed CTLA4 (CD152) is homologous to CD28 but is not expressed onresting T cells and appears following T cell activation (Brunet, J. F.et al. (1987) Nature 328:267-270). CTLA4 appears to be critical innegative regulation of T cell responses (Waterhouse et al. (1995)Science 270:985). Blockade of CTLA4 has been found to remove inhibitorysignals, while aggregation of CTLA4 has been found to provide inhibitorysignals that downregulate T cell responses (Allison and Krummel (1995)Science 270:932). The B7 molecules have a higher affinity for CTLA4 thanfor CD28 (Linsley, P. S. et al. (1991) J. Exp. Med 174:561-569) and B7-1and B7-2 have been found to bind to distinct regions of the CTLA4molecule and have different kinetics of binding to CTLA4 (Linsley et al.(1994) Immunity 1:793).

In the past, reports of the existence of additional members of the B7costimulatory family have been controversial. The antibody BB-1,appeared to recognize a subset of cells greater than either B7-1 or B7-2positive cells, arguing for the existence of another B7-family member,B7-3. The identity of B7-3 had been in part thought to be answered byexpression cloning of T-cell receptor invariant chain using the BB-1antibody. Although invariant chain is not related to the B7 family, thismolecule facilitated a low degree of costimulation when assessed by Tcell proliferation assays.

Very recently, a novel surface receptor termed ICOS was described whichhad sequence identity with CD28 (24%) and CTLA4 (17%) (Hutloff et al.(1999) Nature 397:263; WO 98/38216). Unlike CD28, ICOS was shown to beupregulated on stimulated T cells and caused the secretion of a panel ofcytokines distinct from those mediated by CD28 costimulation (Hutloff etal. (1999) Nature 397:263).

The importance of the B7:CD28/CTLA4 costimulatory pathway has beendemonstrated in vitro and in several in vivo model systems. Blockade ofthis costimulatory pathway results in the development of antigenspecific tolerance in murine and human systems (Harding, F. A. et al.(1992) Nature 356:607-609; Lenschow, D. J. et al. (1992) Science257:789-792; Turka, L. A. et al. (1992) Proc. Natl. Acad. Sci. USA89:11102-11105; Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA90:6586-6590; Boussiotis, V. et al. (1993) J. Exp. Med. 178:1753-1763).Conversely, expression of B7 by B7 negative murine tumor cells inducesT-cell mediated specific immunity accompanied by tumor rejection andlong lasting protection to tumor challenge (Chen, L. et al. (1992) Cell71:1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science259:368-370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:5687-5690.). Therefore, manipulation of the costimulatory pathwaysoffers great potential to stimulate or suppress immune responses inhumans.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules and polypeptides encoded by such nucleicacid molecules, referred to herein as GL50 molecules. Preferred GL50molecules include antigens on the surface of professional antigenpresenting cells (e.g., B lymphocytes, monocytes, dendritic cells,Langerhan cells) and other antigen presenting cells (e.g.,keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes), which costimulate T cell proliferation, bind tocostimulatory receptors ligands on T cells (e.g., CD28, CTLA4, and/orICOS) and/or are bound by antibodies which recognize B7 family members,e.g., anti-GL50 antibodies.

The GL50 nucleic acid and polypeptide molecules of the present inventionare useful, e.g., in modulating the immune response. Accordingly, in oneaspect, this invention provides isolated nucleic acid molecules encodingGL50 polypeptides, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of GL50-encoding nucleicacids.

In one embodiment, a GL50 nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or moreidentical to a nucleotide sequence (e.g., to the entire length of thenucleotide sequence) including SEQ ID NO:1, 3, or 5, or a complementthereof.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown SEQ ID NO:1, 3, or 5, or a complementthereof. In another preferred embodiment, an isolated nucleic acidmolecule of the invention encodes the amino acid sequence of a GL50polypeptide.

Another embodiment of the invention features nucleic acid molecules,preferably the GL50 nucleic acid molecules, which specifically detectthe GL50 nucleic acid molecules relative to nucleic acid moleculesencoding non-GL50 polypeptides. For example, in one embodiment, such anucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides inlength and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, 3, or5, or a complement thereof.

In other preferred embodiments, nucleic acid molecules of the inventionencode naturally occurring allelic variants of a human GL50 polypeptide,wherein the nucleic acid molecules hybridize to a nucleic acid moleculewhich includes SEQ ID NO:1, 3, or 5 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a GL50 nucleic acid molecule, e.g., thecoding strand of a GL50 nucleic acid molecule.

Another aspect of the invention provides a vector comprising a GL50nucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing a polypeptide, preferably a GL50polypeptide, by culturing in a suitable medium, a host cell, e.g., amammalian host cell such as a non-human mammalian cell, of the inventioncontaining a recombinant expression vector, such that the polypeptide isproduced.

Another aspect of this invention features isolated or recombinant GL50polypeptides and proteins.

In one embodiment, the isolated polypeptide is a human GL50 polypeptide.

In yet another embodiment, the isolated GL50 polypeptide is a solubleGL50 polypeptide.

In a further embodiment, the isolated GL50 polypeptide is expressed onthe surface of a cell, e.g., has a transmembrane domain.

In a further embodiment, the isolated GL50 polypeptide plays a role incostimulating the cytokine secretion and/or proliferation of activated Tcells. In another embodiment, the isolated GL50 polypeptide is encodedby a nucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, or 5.

Another embodiment of the invention features an isolated polypeptide,preferably a GL50 polypeptide, which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to a nucleotidesequence (e.g., to the entire length of the nucleotide sequence)including SEQ ID NO:1, 3, or 5 or a complement thereof.

Another embodiment of the invention features an isolated polypeptide,preferably a GL50 polypeptide, which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more identity to an amino acidsequence (e.g., to the entire length of the amino acid sequence)including SEQ ID NO:2, 4, or 6.

This invention further features an isolated GL50 polypeptide which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, or 5, ora complement thereof.

The polypeptides of the present invention can be operatively linked to anon-GL50 polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind polypeptidesof the invention, preferably GL50 polypeptides. In addition, the GL50polypeptides, e.g., biologically active polypeptides, can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a GL50 nucleic acid molecule or polypeptide in abiological sample by contacting the biological sample with an agentcapable of detecting a GL50 nucleic acid molecule or polypeptide suchthat the presence of a GL50 nucleic acid molecule or polypeptide isdetected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of GL50 activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofGL50 polypeptide activity such that the presence of the GL50 polypeptideactivity is detected in the biological sample.

In another aspect, the invention provides a method for modulating GL50polypeptide activity comprising contacting a cell capable of expressingGL50 polypeptide with an agent that modulates GL50 activity such thatthe GL50 activity in the cell is modulated. In one embodiment, the agentinhibits GL50 activity. In another embodiment, the agent stimulates GL50activity. In one embodiment, the agent is an antibody that binds,preferably specifically, to a GL50 polypeptide. In another embodiment,the agent modulates expression of GL50 by modulating transcription of aGL50 gene or translation of a GL50 mRNA. In yet another embodiment, theagent is a nucleic acid molecule having a nucleotide sequence that isantisense to the coding strand of a GL50 mRNA or a GL50 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder (characterized by aberrant GL50polypeptide or nucleic acid expression or activity) or a condition thatwould benefit from modulation, either up or downmodulation, of a GL50molecule by administering an agent which is a GL50 modulator to thesubject. In one embodiment, the GL50 modulator is a GL50 polypeptide. Inanother embodiment the GL50 modulator is a GL50 nucleic acid molecule.In another embodiment a GL50 modulator molecule that modulates theinteraction between GL50 and a ligand of GL50 or a molecule thatinteracts with the intracellular domain of GL50. In yet anotherembodiment, the GL50 modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant GL50 polypeptide or nucleic acid expression is an immune systemdisorder or condition that would benefit from modulation of a GL50activity.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aGL50 polypeptide; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a GL50 polypeptide, wherein awild-type form of the gene encodes a polypeptide with a GL50 activity.

In another aspect the invention provides a method for identifying acompound that binds to or modulates the activity of a GL50 polypeptide.The method includes providing an indicator composition comprising a GL50polypeptide having GL50 activity, contacting the indicator compositionwith a test compound, and determining the effect of the test compound onGL50 activity in the indicator composition to identify a compound thatmodulates the activity of a GL50 polypeptide.

In another aspect, the invention pertains to nonhuman transgenic animalthat contains cells carrying a transgene encoding a GL50 memberpolypeptide.

In one embodiment, the present invention provides methods for treatingcancer involving administering to a subject suffering from a tumorcomprising administering a stimultory form of a GL50 molecule. In apreferred embodiment, the stimulatory form of a GL50 molecule is asoluble form of GL50 and includes the extracellular domain of acostimulatory molecule. In one embodiment, the costimulatory molecule ismonospecific. In one embodiment, the costimulatory molecule is dimeric.In one embodiment, the costimulatory molecule is bivalent.

In another preferred embodiment, the costimulatory molecule is fused toa second protein or polypeptide which includes a portion of animmunoglobulin molecule (e.g., a portion of an immunoglobulin moleculethat includes cysteine residues; a portion of an immunoglobulin moleculethat includes the hinge, CH2, and CH3 regions of a human immunoglobulinmolecule; or a portion of an immunoglobulin molecule that includes thehinge, CH1, CH2, and CH3 regions of a human immunoglobulin molecule). Inyet another embodiment, the portion of the immunoglobulin molecule hasbeen modified to reduce complement fixation and/or Fc receptor binding.

In yet another aspect, the invention pertains to a method for reducingthe proliferation of a tumor cell comprising contacting an immune cellwith an activating form of a GL50 molecule such that an immune responseto the tumor cell is enhanced and proliferation of the tumor cell isreduced.

In one embodiment, the activating form of a GL50 molecule is a solublepolypeptide comprising the extracellular domain of GL50.

In another embodiment, the activating form of a GL50 molecule is a cellassociated polypeptide comprising the extracellular domain of GL50.

In yet another embodiment, the invention pertains to a method forscreening for a compound which modulates GL50 mediated activation of animmune cell comprising: i) contacting a polypeptide comprising at leastone GL50 polypeptide domain with a test compound and a GL50 bindingpartner and ii) identifying compounds that modulate the interaction ofthe polypeptide with the GL50 binding partner to thereby identifycompounds that modulate GL50 mediated activation of an immune cell.

In one embodiment, the polypeptide comprises a GL50 domain selected fromthe group consisting of: a transmembrane domain, a cytoplasmic domain,and an extracellular domain.

In one embodiment, the domain is a splice variant of a GL50 cytoplasmicdomain.

In one embodiment, the GL50 polypeptide domain comprises at least oneamino acid substitution.

In one aspect, the invention pertains to a method for screening for acompound which modulates signal transduction in an immune cellcomprising contacting an immune cell that expresses a GL50 molecule witha test compound and determining the ability of the test compound tomodulate signal transduction via GL50 to thereby identify a compoundwith modulates a signal in an immune cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the complete nucleotide sequence of murine GL50-1(mGL50-1), based on signal sequence clone (position 1-519) and RecAisolated clone (position 374-2718). Predicted nucleotides encoding asignal sequence are boxed and the hydrophobic transmembrane domain isunderlined.

FIG. 2 shows the nucleotide sequence of murine GL50-2 (mGL50-2) product.

FIG. 3 shows a sequence alignment of mGL50-1 and mGL50-2 product.Sequence divergence occurs at nucleotide 1027 for mGL50-1 and at 960 formGL50-2.

FIG. 4 shows isoform specific RT-PCR of mGL50-1 and mGL50-2.

FIG. 5 shows isoform specific Northern Blot analysis of mGL50-1 andmGL50-2.

FIG. 6 shows the nucleotide sequence of AB014553 RACE product. The boxedregion is an area of divergence between the published AB014553 cDNAsequence and the RACE product. Final nested RACE primer extends fromposition 1 to 22, corresponding to nucleotides 655 to 676.

FIG. 7 shows an alignment of the translated RACE product and thepublished AB014553 cDNA. Divergence occurs at residues 299 of thepublished AB014553 cDNA and residues 123 of the RACE product.

FIG. 8 shows the sequence of human GL50 (hGL50).

FIG. 9 shows hydropathy plot analysis of GL50, merged AB014553 RACEproduct (hGL50), and mouse and human B7-1 and B7-2. Significanthydropathy profiles are seen between GL50 and AB014553.

FIG. 10 shows RT-PCR Southern blot analysis of the published AB014553cDNA and AB014553 RACE products.

FIG. 11 shows northern analysis of multiple human tissue RNA blots. Thecoding sequences of the hGL50/AB014553 were used as probes.

FIG. 12 shows a pileup analysis of hGL50, mGL50-1, hB7-1, mB7-2, hB7-2,mB7-2 in which the signal peptide, Ig-like domains, transmembrane, andcytoplasmic domains are indicated. The Predicted hydrophobictransmembrane residues are underlined and asterisks denote residueswhich contribute to Ig structure. The extracellular cysteines andtryptophans, indicators of Ig structure, are shown in bold.

FIG. 13 shows dendrogram analysis representing genetic distances betweenB7-1, B7-2 and GL50 proteins. Y08823 is the chicken CD80-like proteinand MM867065_(—)1 is the mouse butyrophilin.

FIG. 14 shows results of a GL50 COS transfection study. mGL50-1 wasexpressed in COS cells followed by staining with either ICOS-Ig,CD28-Ig, CTLA4-Ig. Binding of ICOS Ig by cells expressing mGL50-1 wasdetected.

FIG. 15 depicts a schematic diagram of mGL50-1 and mGL50-2. Sequencedivergence, indicated by vertical line, occurs at nucleotide 1027 formGL50-1 and 960 for mGL50-2. The repetitive sequence (hatched box) isfound in the 3′ UTR of mGL50-2 encompassing nucleotides 1349-1554.Dashes and arrowheads represent oligonucleotides used in RT-PCRanalysis. Horizontal lines represent probes used in Northern blotanalysis.

FIG. 16 depicts a protein sequence alignment between mGL50-1, mGL50-2,hGL50, and Y08823. Sequences were aligned with PileUp, and sharedresidues between these molecules are boxed. Letters above sequencesdenote secondary peptide structures as predicted for Y08823 based on thecrystal structure of B7-1. The exon encoding hGL50 cytoplasmic domain 1sequences is indicated by bar labeled Cy-1.

FIG. 17 depicts flow cytometric analysis of ICOS binding to mouse,human, and chicken GL50-related proteins. COS cells transfected withexpression plasmids encoding mGL50-1, mGL50-2, hGL50, and the chickenB7-like protein Y08823 were incubated with mICOS-mIgG2am, hICOS-mIgG2amor mCTLA4-mIgG2am, followed by secondary staining with anti-mouse IgG2abiotin and detection with streptavidin-PE.

FIG. 18 depicts ICOS binding to WEHI 231. Titered amounts ofmICOS-mIgG2am or mCTLA4-mIgG2am were used to stain WEHI 231 cells in thepresence of blocking anti B7-1 and B7-2 antibodies or isotype controls.

FIG. 19 depicts ICOS binding to undifferentiated ES cells. Analysis ofundifferentiated ES cells counter stained with anti-B7-1 andmICOS-mIgG2am reagents resulted in the positive staining for both B7-1and ICOS-ligand.

FIG. 20 depicts immunophenotyping of Balb/c and RAG1 −/− splenocytesubsets. Two dimensional plots of 10,000 stained cells are presented;samples with 50,000 data points are indicated by asterisks. (A) Enrichedsplenocytes from Balb/C or RAG1 −/− mice were stained with mICOS-mIgG2amand FITC-conjugated antibodies against CD3, CD24, CD45R/B220, pan NK,MHC class II, or CD40. To further phenotype the CD4+, ICOS-ligand+cells, RAG1 −/− cells were stained with PE-labeled anti-CD4 andFITC-labeled anti-CD11c. (B) Enriched splenocytes from RAG1 −/− andBalb/C mice (untreated, ConA activated, or LPS activated) were stainedwith mICOS-mIgG2am and antibodies to CD4, CD8, CD19, CD11b, CD11c andCD69.

FIG. 21 depicts a phylogenetic representation of GL50/B7 ligands andCD28/CTLA4/ICOS receptors. Distance proportional phylograms weregenerated using values from Tables 5 (GL50/B7 ligands) and 6(CD28/CTLA4/ICOS). Bars represent genetic distance expressed assubstitutions per 100 amino acids. (A) Phylogram of GL50/B7 relatedproteins. Accession No. MMU67065_(—)1 represents mouse butyrophilin. (B)Phylogram of ICOS/CD28/CTLA4 proteins.

FIG. 22 depicts proliferation and cytokine induction by GL50costimulation of T cells, in the absence or presence of anti-CD28blocking antibodies. Note: hGL50.Fc is the same as hGL50-IgG2am.

FIG. 23 depicts T cell proliferation induced by GL50 costimulation inthe presence of varied concentrations of anti-CD28 blocking antibodiesand anti-CD3 stimulation.

FIG. 24 depicts cytokine induction by GL50 costimulation in T cells inthe absence or presence of CD28 stimulation.

FIG. 25 depicts the ability of GL50-IgG2a to inhibit tumor growth inmice.

FIG. 26 depicts the sequence of the hICOS-mIgG2am fusion protein. (A)The nucleotide sequence encoding hICOS-mIgG2am (set forth as SEQ IDNO:23). The oncostatin-M leader sequence is encoded by the underlinednucleotides. Boxed nucleotides encode the mouse IgG2am domain of thefusion protein. The translation initiation site is indicated by an X.Introns and untranslated regions are indicated by a dashed line. Thestop codon is indicated by a double underline. (B) The predicted aminoacid sequence (set forth as SEQ ID NO:24) of the hICOS-mIgG2am fusionprotein.

FIG. 27 depicts the sequence of the mICOS-mIgG2am fusion protein. (A)The nucleotide sequence encoding mICOS-mIgG2am (set forth as SEQ IDNO:25). The oncostatin-M leader sequence is encoded by the underlinednucleotides. Boxed nucleotides encode the mouse IgG2am domain of thefusion protein. The translation initiation site is indicated by an X.Introns and untranslated regions are indicated by a dashed line. Thestop codon is indicated by a double underline. (B) The predicted aminoacid sequence (set forth as SEQ ID NO:26) of the mICOS-mIgG2am fusionprotein.

FIG. 28 depicts the sequence of the hGL50-mIgG2am fusion protein. (A)The nucleotide sequence encoding hGL50-mIgG2am (set forth as SEQ IDNO:27). The oncostatin-M leader sequence is encoded by the underlinednucleotides. Boxed nucleotides encode the mouse IgG2am domain of thefusion protein. The translation initiation site is indicated by an X.Introns and untranslated regions are indicated by a dashed line. Thestop codon is indicated by a double underline. (B) The predicted aminoacid sequence (set forth as SEQ ID NO:28) of the hGL50-mIgG2am fusionprotein.

FIG. 29 depicts the sequence of the mGL50-mIgG2am fusion protein. (A)The nucleotide sequence encoding mGL50-mIgG2am (set forth as SEQ IDNO:29). The oncostatin-M leader sequence is encoded by the underlinednucleotides. Boxed nucleotides encode the mouse IgG2am domain of thefusion protein. The translation initiation site is indicated by an X.Introns and untranslated regions are indicated by a dashed line. Thestop codon is indicated by a double underline. (B) The predicted aminoacid sequence (set forth as SEQ ID NO:30) of the mGL50-mIgG2am fusionprotein.

FIG. 30 depicts ICOS-Ig staining of various splenic cell types.

FIG. 31 depicts the reduction of tumorigenicity of tumor cellstransfected with GL50.

DETAILED DESCRIPTION OF THE INVENTION

In addition to the previously characterized B lymphocyte activationantigens, e.g., B7-1 and B7-2, there are other antigens on the surfaceof antigen presenting cells (e.g., B cells, monocytes, dendritic cells,Langerhan cells, keratinocytes, endothelial cells, astrocytes,fibroblasts, oligodendrocytes) which costimulate T cells.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as GL50 polypeptides. Murine GL50-1(mGL50-1) was isolated from an IL-12 activated mouse lymph node library.The nucleotide sequence of mGL50-1 is shown in SEQ ID NO:1. The derivedpolypeptide sequence of full length mouse mGL50-1 is shown in SEQ IDNO:2. The sequence shares approximately 20% sequence identity with mouseB7-1 and mouse B7-2. mGL50-1 encodes a 322 amino acid polypeptidecontaining a leader sequence, extracellular Ig-like domains, ahydrophobic transmembrane domain, and an intracellular domain comprisingone tyrosine residue.

3′ RACE PCR with mouse peripheral blood lymphocyte (PBL) RNA revealed analternatively spliced form of mouse GL50 (mGL50-2). The nucleotidesequence of murine GL50-2 (mGL50-2) is shown in SEQ ID NO:3. Thenucleotide sequence encoded a polypeptide having a divergent 27 aminoacid intracellular domain, which included an additional three tyrosines,a 3′ untranslated region with consensus polyadenylation signal, and apoly A tail which are shown in SEQ ID NO:4. Transcripts of both mGL50-1and mGL50-2 were found by RT-PCR and Northern blot analysis and werepredominantly localized in lymphoid organs of multiple tissue panels.The murine GL50 sequences identified were found to be related to apreviously reported human brain cDNA clone, GenBank Accession NumberAB014553.

3′ RACE of human PBL cDNA was performed to identify human clones relatedto murine GL50. Clones encoding alternative 3′ sequences wereidentified. The nucleotide sequence of the resulting human GL50 (hGL50[AB014553-RACE]) clone is shown in SEQ ID NO:5. The nucleotide sequenceencodes a 309 amino acid protein sharing about 26% amino acid sequenceidentity with the mGL50-1, 28% identity with mGL50-2, and amino acidsequence, approximately 13% amino acid sequence identity with humanB7-1, and about 13% amino acid sequence identity with human and mouseB7-2.

Flow cytometric assays using murine GL50-1Ig fusion protein as a reagentdemonstrated binding to COS transfectants expressing mouse ICOS, but notto cells expressing CD28 or CTLA-4. These results confirm that GL50molecules are novel members of the B7 family of molecules.

GL50 Nucleic Acid and Polypeptide Molecules

In one embodiment, the isolated nucleic acid molecules of the presentinvention encode eukaryotic GL50 polypeptides.

The GL50 family of molecules share a number of conserved regions,including signal domains, IgV domains and the IgC domains. For example,in the case of mGL50-1 (SEQ ID NO:1), the consensus 2718 nucleotidemGL50-1 sequence encodes a 322 amino acid protein with a predicted massof 36 kDa. Hydropathy plot of the open reading frame predicted astructure corresponding to a leader sequence (encoded by aboutnucleotides 67 to 195), an extracellular domain (encoded by aboutnucleotides 196 to 904), a hydrophobic transmembrane region (encoded byabout nucleotides 905 to 961) and a potential intracellular cytoplasmicdomain (encoded by about nucleotides 962 to 1032). Signal peptidecleavage was predicted at position 46 in the amino acid sequence. In oneembodiment, the extracellular domain of a GL50 polypeptide comprises theIgV and IgC domains after cleavage of the signal sequence, but not thetransmembrane and cytoplasmic domains of a GL50 polypeptide (e.g.,corresponding to the amino acid sequence from about amino acid 47-277 ofGL50-1 or the amino acid sequence from about amino acid 22 to aboutamino acid 278 of hGL50 as set forth in FIG. 16).

Analysis of the mGL50-1 amino acid sequence suggested structuralsimilarity to an Ig-domain in the cytoplasmic domain of the protein. Inkeeping with an Ig-like structure, 4 cysteines were found in theextracellular domain, allowing for the possibility of intramolecularbonding and distinct structural conformation corresponding to anIgV-like domain and an IgC-like domain. These regions are both Igsuperfamily member domains and are art recognized. These domainscorrespond to structural units that have distinct folding patterns knownas Ig folds. Ig folds are comprised of a sandwich of two β sheets, eachconsisting of antiparallel β strands of 5-10 amino acids with aconserved disulfide bond between the two sheets in most, but not all,domains. IgC domains of Ig, TCR, and MHC molecules share the same typesof sequence patterns and are referred to as C₁-set within the Igsuperfamily. Other IgC domains fall within other sets. IgV domains alsoshare sequence patterns and are called V set domains. IgV domains arelonger than C-domains and form an additional pair of β strands.

An alignment of the mGL50-2, mGL50-1, hGL50, and chicken Y08823 moleculeare presented in FIG. 16. Each of the molecules comprises a signalpeptide, an IgV-like domaine, an IgC-like domain, a transmembranedomaine and a cytoplasmic domain. Domains of mGL50-2, hGL50, and Y08823corresponding to those in mGL50-1 are presented in FIG. 16.

A protein alignment was made of the GL50 polypeptides, the publishedAB014553 sequence, and the human and mouse B7-1 and B7-2 sequences usingthe Geneworks protein alignment program with the parameters set at: costto open gap=5, cost to lengthen gap=5, minimum diagonal length=4,maximum diagonal offset=130, consensus cutoff=50%, and using the Pam 250matrix. The results of the alignment are presented below in Table 1.TABLE 1 Protein Alignment for G150-related proteins AB014553 hGL50mGL50-1 mGL50-2 hB7-2 mB7-2 hB7-1 mB7-1 ABO14553 100 59 26 28 13 13 13 7hGL50 100 42 41 17 17 17 12 GL50-1 100 92 19 19 20 14 GL50-2 100 20 2120 13 hB7-2 100 48 19 21 mB7-2 100 20 24 hB7-1 100 41 mB7-1 100Alignments were done using the Geneworks protein alignment program withthe cost to open gap = 5, cost to lengthen gap = 5, min. diagonal length= 4, max. diagonal offset = 130, consensus cutoff = 50%, Pam 250 matrix.

Table 1 shows that the hGL50 polypeptide has approximately 59% aminoacid sequence identity with the polypeptide encoded by AB014553 andapproximately 40% amino acid sequence identity with mGL50-1 and mGL50-2.mGL50-1 and mGL50-2 share a higher degree of amino acid sequenceidentity, approximately 92%. The GL50 polypeptides share approximately20% amino acid sequence identity with other B7 family molecules.

Another alignment was made to determine the extent of relatednessbetween murine GL50, hGL50, human B7-1, mouse B7-1, mouse B7-2, andhuman B7-2 protein sequences. Using a Pileup analysis (FIG. 12), 18amino acid locations aligned identically between all six moleculeswithin the extracellular domain. Of the 32 positions that define thepredicted IgV-like and IgC-like folds of the B7 molecule, 13 areidentically conserved between all six molecules, most notably the 4cysteines that allow intramolecular folding of domains. Other areas ofsignificant sequence conservation were also seen in the extracellulardomain, but interestingly the identities of GL50 sequences in certainlocations aligned more closely with either B7-1 or B7-2 (identity scoreof 8). For example, a valine residue corresponding to position 86 ofmGL50-1 is shared by hGL50, and B7-2 sequences, but not B7-1. Likewise,the tyrosine at position 87 of mouse mGL50-1 is conserved atcorresponding locations in hGL50 and B7-1, but not B7-2. Of the 16positions with identity scores of 8, 5 positions are shared by mousemGL50-1/hGL50 and B7-1, 4 positions are shared between mousemGL50-1/hGL50 and B7-2, and 6 positions are shared between B7-1 andB7-2. Based on the peptide structure, these results suggest that theGL50 sequences occupy a phylogenetic space parallel to the B7 family ofproteins.

Molecular phylogeny analysis (GrowTree) measuring genetic distance interms of substitutions per 100 amino acids resulted in a dendrogram(FIG. 13) with independent clustering of mouse/hGL50 (85), m/hB7-2(68)and m/hB7-1 (88). As an outgroup, mmu67065_(—)1 (mouse butyrophilin) wasused. The chicken clone Y08823 also was found to be more closely alignedwith the GL50 sequences (˜140) than the B7sequences (215-320),indicating that these sequences comprised a distinct subfamily ofproteins. Distances between the GL50, B7-2 and B7-1 branches were high(216-284), suggesting that large numbers of substitutions have occurredbetween these molecules since the inception of the human and rodentlineage. The genetic distances among the GL50 nucleic acid molecules arepresented below in Table 2. TABLE 2 Genetic Distances among B7 familymembers hGL50 mGL50-1 YO8823 hB7-2 mB7-2 hB7-1 mB7-1 mmu67065_1 hGL50 085 142 284 263 226 260 188 mGL50-1 0 139 225 216 229 257 223 YO8823 0235 322 215 223 223 hB7-2 0 68 222 190 215 mB7-2 0 88 211 21 bB7-1 0 88211 mB7-1 0 271 mmu67065_1 0

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Definitions

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells. The term “antigen presenting cell” includesprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, Langerhans cells) as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Tcell responses, e.g., cytokine production, and cellular cytotoxicity. Inaddition, the term immune response includes immune responses that areindirectly effected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages.

As used herein, the term “costimulatory receptor” includes receptorswhich transmit a costimulatory signal to a immune cell, e.g., CD28. Asused herein, the term “inhibitory receptors” includes receptors whichtransmit a negative signal to an immune cell (e.g., CTLA4). Aninhibitory signal as transduced by an inhibitory receptor can occur evenif a costimulatory receptor (such as CD28) in not present on the immunecell and, thus, is not simply a function of competition betweeninhibitory receptors and costimulatory receptors for binding ofcostimulatory molecules (Fallarino et al. (1998) J. Exp. Med 188:205).Transmission of an inhibitory signal to an immune cell can result inunresponsiveness or anergy or programmed cell death in the immune cell.Preferably transmission of an inhibitory signal operates through amechanism that does not involve apoptosis. As used herein the term“apoptosis” includes programmed cell death which can be characterizedusing techniques which are known in the art. Apoptotic cell death can becharacterized, e.g., by cell shrinkage, membrane blebbing and chromatincondensation culminating in cell fragmentation. Cells undergoingapoptosis also display a characteristic pattern of internucleosomal DNAcleavage.

In addition to differences in types of receptors, different forms ofcostimulaotry molecules can be either activating or inhibitory. Forexample, in the case of an activating receptor a signal can betransmitted e.g., by a multivalent form of a costimulatory molecule thatresults in crosslinking of an activating receptor, or a signal can beinhibited, e.g., by a form of a costimulatory molecule that binds to anactivating receptor, but fails to transmit an activating signal, e.g.,by competing with activating forms of costimulatory molecules forbinding to the receptor. (Certain soluble forms of costimulatorymolecules can be inhibitory, however, there are instances in which asoluble molecule can be stimulatory). Similarly, depending upon the formof costimulatory molecule that binds to an inhibitory receptor, either asignal can be transmitted (e.g., by a multivalent form of acostimulatory molecule that results in crosslinking of an activatingreceptor) or a signal can be inhibited (e.g., by a form of acostimulatory molecule that binds to an inhibitory receptor, but failsto transmit an inhibitory signal). The effects of the various modulatoryagents can be easily demonstrated using routine screening assays asdescribed herein.

As used herein, the term “costimulate” with reference to activatedimmune cells includes the ability of a “costimulatory molecule” toprovide a second, non-activating receptor mediated signal (a“costimulatory signal”) that induces proliferation or effector function.For example, a costimulatory signal can result in cytokine secretion,e.g., in a T cell that has received a T cell-receptor-mediated signal.Immune cells that have received a cell-receptor mediated signal, e.g.,via an activating receptor are referred to herein as “activated immunecells.”

As used herein, the term “activating receptor” includes immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), or bind to antibodies. Such activating receptors includeT cell receptors (TCR), B cell receptors (BCR), cytokine receptors, LPSreceptors, complement receptors, and Fc receptors.

For example, T cell receptors are present on T cells and are associatedwith CD3 molecules. T cell receptors are stimulated by antigen in thecontext of MHC molecules (as well as by polyclonal T cell activatingreagents). T cell activation via the TCR results in numerous changes,e.g., protein phosphorylation, membrane lipid changes, ion fluxes,cyclic nucleotide alterations, RNA transcription changes, proteinsynthesis changes, and cell volume changes.

As used herein, the term “inhibitory signal” refers to a signaltransmitted via an inhibitory receptor (e.g., CTLA4) on a immune cell.Such a signal antagonizes a signal via an activating receptor (e.g., viaa TCR, CD3, BCR, or Fc molecule) and can result in, e.g., inhibition ofsecond messenger generation; an inhibition of proliferation; aninhibition of effector function in the immune cell, e.g., reducedphagocytosis, reduced antibody production, reduced cellularcytotoxicity, the failure of the immune cell to produce mediators, (suchas cytokines (e.g., IL-2) and/or mediators of allergic responses); orthe development of anergy.

As used herein, the term “adjuvant” includes agents which potentiate theimmune response to an antigen (e.g., a tumor-associated antigen).Adjuvants can be administered in conjunction with costimulatorymolecules to additionally augment the immune response.

As used herein, the term “monospecific” includes molecules which haveonly one specificity, i.e., they specifically bind to their cognateligand, e.g., CD28, CTLA4, or ICOS on T cells. Such monospecific agentshave not been engineered to include additional specificities and, thus,do not bind in a targeted manner to other cell surface molecules. Asused herein the term “oligospecific” includes molecules having more thanone specificity, e.g., having an additional specificity for a moleculeother than afor their cognate ligand, e.g., a specificity for a cellsurface molecule, such as a tumor associated antigen or a T cellreceptor. As used herein, the term “bivalent” includes solublecostimulatory molecules that have two binding sites for interaction withtheir ligand per molecule. As used herein, the term “dimeric” includesforms that are present as homodimers, i.e., as a unit comprised of twoidentical subunits which are joined together, e.g., by disulfide bonds.As used herein, the term “multimeric” includes soluble forms having morethan two subunits.

In another embodiment, an activating form of a GL50 molecule is asoluble GL50 molecule. As used herein, the term “soluble” includesmolecules, e.g., costimulatory molecules, which are not cell associated.Soluble costimulatory molecules retain the function of the cellassociated molecules from which they are derived, e.g., they are capableof binding to their cognate ligands on T cells and mediating signaltransduction via a CD28 and/or CTLA4 molecule on a T cell, however, theyare in soluble form, i.e., are not membrane bound. Preferably, thesoluble compositions comprise an extracellular domain of a costimulatorymolecule.

Preferably, such a soluble form of a GL50 comprises at least a portionof the extracellular domain of a GL50 molecule. As used herein, the term“extracellular domain of a GL50 molecule” includes a portion of a GL50molecule which, in the cell-associated form of the GL50 molecule, isextracellular. Preferably, the extracellular domain is the extracellulardomain of a human GL50 molecule. In one embodiment, a solublecostimulatory molecule comprises an extracellular domain of a GL50molecule and further comprises a signal sequence.

As used herein, the term “unresponsiveness” includes refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or exposure to high doses of antigen. Asused herein, the term “anergy” or “tolerance” includes refractivity toactivating receptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory molecule) resultsin failure to produce cytokines and, thus, failure to proliferate.Anergic T cells can, however, mount responses to unrelated antigens andcan proliferate if cultured with cytokines (e.g., IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the API sequence that canbe found within the enhancer (Kang et al. (1992) Science 257:1134).

The GL50 polypeptide and nucleic acid molecules comprise a family ofmolecules having certain conserved structural and functional features.The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin. Members of a family may also have commonfunctional characteristics.

The GL50 molecules described herein are members of a larger family ofmolecules, the B7 family of costimulatory molecules. The term “B7family” or “B7 molecules” as used herein includes costimulatorymolecules that share sequence homology with B7 polypeptides, e.g., withB7-1, B7-2, B7-3 (recognized by the antibody BB-1), and/or GL50. Forexample, as shown in Table 1 above, human B7-1 and human B7-2 shareapproximately 20% amino acid sequence identity. In addition, the B7family of molecules share a common function, e.g., the ability to bindto a B7 family ligand (e.g., one or more of CD28, CTLA4, or ICOS) and/orther ligands on immune cells and have the ability to inhibit or inducecostimulation of immune cells.

As used herein, the term “activity” with respect to a GL50 polypeptideincludes activities which are inherent in the structure of a GL50polypeptide. The term “activity” includes the ability to modulate acostimulatory signal in activated T cells and induce proliferationand/or cytokine secretion. In addition, the term “activity” includes theability of a GL50 polypeptide to bind its natural ligand or bindingpartner. Preferably, the ligand to which a GL50 polypeptide binds is anICOS molecule. As used herein “activating forms” of costimulatorymolecules transmit a signal via a costimulatory receptor (e.g., a signalwhich activates an immune cell if the receptor is an inhibitory receptorwhich transmits a costimulatory signal (e.g., CD28 or ICOS) or aninhibitory signal if the receptor is one which transmits a negativesignal to an immune cell (e.g., CTLA4). Inhibitory forms of acostimulatory molecule prevent transmission of a signal to an immunecell (e.g., either a costimulatory signal or a negative signal).

As used herein, the term “tumor” includes both benign and malignant(cancerous) neoplasias, (e.g., carcinomas, sarcomas, leukemias, andlymphomas). The term “cancer” includes primary malignant tumors (e.g.,those whose cells have not migrated to sites in the subject's body otherthan the site of the original tumor) and secondary malignant tumors(e.g., those arising from metastasis, the migration of tumor cells tosecondary sites that are different from the site of the original tumor).

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid molecule comprises anucleotide sequence which is complementary to a “sense” nucleic acidmolecule encoding a protein, e.g., complementary to the coding strand ofa double-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., GL50). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778).Such single chain antibodies are also intended to be encompassed withinthe term “antigen-binding portion” of an antibody. Any VH and VLsequences of specific scFv can be linked to human immunoglobulinconstant region cDNA or genomic sequences, in order to generateexpression vectors encoding complete IgG molecules or other isotypes. VHand VI can also be used in the generation of Fab, Fv or other fragmentsof immunoglobulins using either protein chemistry or recombinant DNAtechnology. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof, e.g. humanized, chimeric, etc.Preferably, antibodies of the invention bind specifically orsubstantially specifically to GL50 molecules. The terms “monoclonalantibodies” and “monoclonal antibody composition”, as used herein, referto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of an antigen, whereas the term “polyclonal antibodies” and“polyclonal antibody composition” refer to a population of antibodymolecules that contain multiple species of antigen binding sites capableof interacting with a particular antigen. A monoclonal antibodycomposition, typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds GL50 is substantially free of antibodies that specifically bindantigens other than GL50). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

As used herein, “binding partner” is a target molecule or a moleculewith which a GL50 polypeptide binds or interacts in nature (e.g., aligand or an intracellular interactor molecule (such as a molecule thatacts either upstream or downstream of GL50 in a signal transductionpathway)), such that a GL50 activity is achieved.

The term “signal transduction” is intended to encompass the processingof physical or chemical signals from the extracellular environmentthrough the cell membrane and into the cell, and may occur through oneor more of several mechanisms, such as activation/inactivation ofenzymes (such as proteases, or other enzymes which may alterphosphorylation patterns or other post-translational modifications),activation of ion channels or intracellular ion stores, effector enzymeactivation via guanine nucleotide binding protein intermediates,formation of inositol phosphate, activation or inactivation of adenylylcyclase, direct activation (or inhibition) of a transcriptional factorand/or activation. A “signaling pathway” refers to the componentsinvolved in “signal transduction” of a particular signal into a cell.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid molecule and the aminoacid sequence encoded by that nucleic acid molecule, as defined by thegenetic code. GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine(Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AATAspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid(Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC,GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATTLeucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination (end)TAA, TAG, TGA signalAn important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAmolecule coding for a GL50 polypeptide of the invention (or any portionthereof) can be used to derive the GL50 amino acid sequence, using thegenetic code to translate the DNA or RNA molecule into an amino acidsequence. Likewise, for any GL50-amino acid sequence, correspondingnucleotide sequences that can encode GL50 polypeptide can be deducedfrom the genetic code (which, because of its redundancy, will producemultiple nucleic acid sequences for any given amino acid sequence).Thus, description and/or disclosure herein of a GL50 nucleotide sequenceshould be considered to also include description and/or disclosure ofthe amino acid sequence encoded by the nucleotide sequence. Similarly,description and/or disclosure of a GL50 amino acid sequence hereinshould be considered to also include description and/or disclosure ofall possible nucleotide sequences that can encode the amino acidsequence.

II. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode GL50 polypeptides or biologically active portions thereof,as well as nucleic acid fragments sufficient for use as hybridizationprobes to identify GL50-encoding nucleic acid molecules (e.g., GL50mRNA) and fragments for use as PCR primers for the amplification ormutation of GL50 nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. For example, with regards to genomic DNA, the term“isolated” includes nucleic acid molecules which are separated from thechromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid molecule is free of sequenceswhich naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid molecule is derived. For example, in variousembodiments, the isolated GL50 nucleic acid molecule can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. An “isolated”GL50 nucleic acid molecule may, however, be linked to other nucleotidesequences that do not normally flank the GL50 sequences in genomic DNA(e.g., the GL50 nucleotide sequences may be linked to vector sequences).In certain preferred embodiments, an “isolated” nucleic acid molecule,such as a cDNA molecule, also may be free of other cellular material.However, it is not necessary for the GL50 nucleic acid molecule to befree of other cellular material to be considered “isolated” (e.g., aGL50 DNA molecule separated from other mammalian DNA and inserted into abacterial cell would still be considered to be “isolated”).

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, or 5, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example,using all or portion of the nucleic acid sequence of SEQ ID NO:1, 3, or5, as a hybridization probe, GL50 nucleic acid molecules can be isolatedusing standard hybridization and cloning techniques (e.g., as describedin Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, or 5 can be isolated by the polymerase chain reaction (PCR)using synthetic oligonucleotide primers designed based upon the sequenceof SEQ ID NO:1, 3, or 5, respectively.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to GL50 nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, 3, or5.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, 3, or 5, or a portion of anyof these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, or 5,is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:1, 3, or 5, respectively, such that it can hybridizeto the nucleotide sequence shown in SEQ ID NO:1, 3, or 5, respectively,thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore homologous to the nucleotide sequence (e.g., to the entire lengthof the nucleotide sequence) shown in SEQ ID NO:1, 3, or 5, or a portionof any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, or 5, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of a GL50 polypeptide. Thenucleotide sequence determined from the cloning of the GL50 genes allowsfor the generation of probes and primers designed for use in identifyingand/or cloning other GL50 family members, as well as GL50 familyhomologues from other species. The probe/primer typically comprises asubstantially purified oligonucleotide. In one embodiment, theoligonucleotide comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, 75, or 100 consecutive nucleotides of a sense sequence of SEQ IDNO:1, 3, or 5, or of a naturally occurring allelic variant or mutant ofSEQ ID NO:1, 3, or 5. In another embodiment, a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at leastabout 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 1000, or 1100nucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule of SEQ ID NO:1, 3, or 5.

In another embodiment, a nucleic acid molecule of the inventioncomprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, or 1100 contiguous nucleotides of SEQ ID NO:1, 3, or 5.

In one embodiment, a nucleic acid molecule of the invention, e.g., foruse as a probe, does not include the portion of SEQ ID NO:1 from aboutnucleotides 1-370 of SEQ ID NO:5.

Preferably, an isolated nucleic acid molecule of the invention comprisesat least a portion of the coding region of SEQ ID NO:1 (shown innucleotides 67-1032) or SEQ ID NO:3 (shown in nucleotides 1-1041) or SEQID NO:5 (shown in nucleotides 24-950). In another embodiment, a nucleicacid molecule of the invention comprises the entire coding region of SEQID NO:1, 3, or 5.

In other embodiments, a nucleic acid molecule of the invention has atleast 70% identity, more preferably 80% identity, and even morepreferably 90% identity with a nucleic acid molecule comprising: atleast about 300, 400, 500, 600, 700, 800, or at about 900 nucleotides ofSEQ ID NO:1, 3, or 5, or at least about 1000 or 1100 contiguousnucleotides of SEQ ID NO:1 or 3.

Probes based on the GL50 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissues which misexpress a GL50 polypeptide, such as by measuring alevel of a GL50-encoding nucleic acid in a sample of cells from asubject e.g., detecting GL50 mRNA levels or determining whether agenomic GL50 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aGL50 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1, 3, or 5, which encodes a polypeptidehaving a GL50 biological activity (the biological activities of the GL50polypeptides are described herein), expressing the encoded portion ofthe GL50 polypeptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the GL50 polypeptide.

Nucleic acid molecules that differ from SEQ ID NO:1, 3, or 5 due todegeneracy of the genetic code, and thus encode the same a GL50 memberprotein as that encoded by SEQ ID NO:1, 3, or 5 are encompassed by theinvention. Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence shown in SEQ ID NO:2, 4 or 6. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a GL50 polypeptide.

In addition to the GL50 nucleotide sequences shown in SEQ ID NO:1, 3, or5, it will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theGL50 polypeptides may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the GL50 genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a GL50polypeptide, preferably a mammalian GL50 polypeptide, and can furtherinclude non-coding regulatory sequences, and introns. Such naturalallelic variations include both functional and non-functional GL50polypeptides and can typically result in 1-5% variance in the nucleotidesequence of a GL50 gene. Any and all such nucleotide variations andresulting amino acid polymorphisms in GL50 genes that are the result ofnatural allelic variation and that do not alter the functional activityof a GL50 polypeptide are intended to be within the scope of theinvention.

Moreover, nucleic acid molecules encoding other GL50 family members and,thus, which have a nucleotide sequence which differs from the GL50family sequences of SEQ ID NO:1, 3, or 5 are intended to be within thescope of the invention. For example, another mGL50-1 can be identifiedbased on the nucleotide sequence of hGL50. Moreover, nucleic acidmolecules encoding GL50 polypeptides from different species, and thuswhich have a nucleotide sequence which differs from the GL50 sequencesof SEQ ID NO:1, 3, or 5 are intended to be within the scope of theinvention. For example, an ortholog of the mGL50-1 can be identifiedbased on the murine nucleotide sequence.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the GL50 molecules of the invention can be isolated, e.g.,based on their homology to the GL50 nucleic acids disclosed herein usingthe cDNAs disclosed herein, or portions thereof, as hybridization probesaccording to standard hybridization techniques. For example, a GL50 DNAcan be isolated from a human genomic DNA library using all or portion ofSEQ ID NO:1, 3, or 5 as a hybridization probe and standard hybridizationtechniques (e.g., as described in Sambrook, J., et al. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid moleculeencompassing all or a portion of a GL50 gene can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon the sequence of SEQ ID NO:1, 3, or 5. For example, mRNA can beisolated from cells (e.g., by the guanidinium-thiocyanate extractionprocedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNAcan be prepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned based upon the nucleotide sequence shown in SEQ ID NO:1, 3, or5. A nucleic acid molecule of the invention can be amplified using cDNAor, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a GL50 nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention can be identified based on shared nucleotide sequence identityusing a mathematical algorithm. Such algorithms are outlined in moredetail below (see, e.g., section III).

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 15, 20, 25, 30 or more nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, or 5. In otherembodiment, the nucleic acid molecule is at least 30, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, or 600 nucleotides in length. As usedherein, the term “hybridizes under stringent conditions” is intended todescribe conditions for hybridization and washing under which nucleotidesequences at least 30%, 40%, 50%, or 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 70%, more preferably at leastabout 80%, even more preferably at least about 85% or 90% homologous toeach other typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1, 3, or 5 corresponds to anaturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). In addition to the GL50nucleotide sequences shown in SEQ ID NO:1, 3, or 5 it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to minor changes in the nucleotide or amino acid sequences ofa GL50 may exist within a population. Such genetic polymorphism in aGL50 gene may exist among individuals within a population due to naturalallelic variation. Such natural allelic variations can typically resultin 1-2% variance in the nucleotide sequence of the gene. Such nucleotidevariations and resulting amino acid polymorphisms in a GL50 that are theresult of natural allelic variation and that do not alter the functionalactivity of a GL50 polypeptide are within the scope of the invention.

In addition to naturally-occurring allelic variants of GL50 sequencesthat may exist in the population, the skilled artisan will furtherappreciate that minor changes may be introduced by mutation intonucleotide sequences, e.g., of SEQ ID NO:1, 3, or 5, thereby leading tochanges in the amino acid sequence of the encoded protein, withoutaltering the functional activity of a GL50 polypeptide. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues may be made in the sequence of SEQID NO:1, 3, or 5. A “non-essential” amino acid residue is a residue thatcan be altered from the wild-type sequence of a GL50 nucleic acidmolecule (e.g., the sequence of SEQ ID NO:1, 3, or 5) without alteringthe functional activity of a GL50 molecule. Exemplary residues which arenon-essential and, therefore, amenable to substitution, can beidentified by one of ordinary skill in the art by performing an aminoacid alignment of B7 family members (or of GL50 family members) anddetermining residues that are not conserved. Such residues, because theyhave not been conserved, are more likely amenable to substitution.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding GL50 polypeptides that contain changes in amino acidresidues that are not essential for a GL50 activity. Such GL50polypeptides differ in amino acid sequence from SEQ ID NO:2, 4, or 6 yetretain an inherent GL50 activity. An isolated nucleic acid moleculeencoding a non-natural variant of a GL50 polypeptide can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 3, or 5 such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced into SEQ ID NO:1,3, or 5 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more non-essential amino acid residues.A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a GL50 is preferably replaced with another amino acid residuefrom the same side chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a GL50 coding sequence, such as bysaturation mutagenesis or rational cassette mutagenesis, and theresultant mutants can be screened for their ability to bind to a ligand,or to bind to intracellular interactor molecules to identify mutantsthat retain functional activity. Following mutagenesis, the encoded GL50mutant protein can be expressed recombinantly in a host cell and thefunctional activity of the mutant protein can be determined using assaysavailable in the art for assessing a GL50 activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding GL50 polypeptides that contain changes in amino acidresidues that are not essential for activity. Homology alignments, suchas the pile-up analysis shown herein, can be used to select amino acidswhich may be amenable to alteration. For example, the 18 amino acidlocations which aligned identically between all six molecules within theextracellular domain are well conserved and are, therefore, less likelyto be amenable to alteration. Similarly, of the 32 positions that definethe predicted IgV-like and IgC-like folds of the B7 family molecules, 13are identically conserved between all six molecules, most notably the 4cysteines that allow intramolecular folding of domains. Therefore, theseamino acids are unlikely to be amenable to alteration. Other areas ofsignificant sequence conservation were also seen in the extracellulardomain. For example, valine residue corresponding to position 86 ofmGL50-1 is shared by hGL50, and B7-2 sequences may not be amenable toalteration. Likewise, the tyrosine at position 87 of mouse mGL50-1 whichis conserved at corresponding locations in hGL50 and B7-1. The 16positions with identity scores of 8 (5 positions are shared by mousemGL50-1/hGL50 and B7-1, 4 positions shared between mouse mGL50-1/hGL50and B7-2, and 6 positions are shared between B7-1 and B7-2) may not beamenable to alteration. In addition, positions in the transmembraneand/or cytoplasmic domains conserved among the GL50 family members (inparticular tyrosind residues in the transmembrane or cytoplasmic domainof a GL50 molecule). Again, these positions are unlikely to be amenableto alteration if GL50 activity is to be maintained.

Yet another aspect of the invention pertains to non-naturally occurringGL50 molecules nucleic acid molecules which are chimeric in that theycomprise a nucleic acid sequence encoding GL50 transmembrane orcytoplasmic domain which they do not naturally comprise. For example, inone embodiment, transmembrane and/or cytoplasmic domains of a GL50domain can be “swapped” or “shuffled” using standard molecular biologytechniques to create GL50 molecules that have altered signaltransduction properties as compared to a naturally occurring GL50molecule. Such nucleic acid and polypeptide molecules are also embracedby the invention.

In yet another aspect, GL50 nucleic acid molecules can be engineered tocomprise nucleic acid sequences encoding at least a portion of anotherB7 family member, e.g., B7-1 or B7-2. For example, using standardtechniques, nucleic acid molecules can be made that encode hybridGL50/B7 molecules with ligand binding and/or signaling properties thatdiffer from those seen in naturally occurring molecules. For example, inone embodiment, the sequence of chicken GL50 (Y08823) can be used todesign molecules with altered signaling and/or binding properties. Thesequence similarity between avian GL50 and mammalian forms of themolecule and their difference in ligand preference can be exploited tothis end. For instance, progressive substitution of residues conservedbetween avian GL50-like protein (Y08823) and GL50 with those found inGL50 (to make the molecule more GL50-like) may result in a functionalmolecule that binds to ICOS and CD28 and CTLA4. Ig-fusion or otherconstructs comprising huybrid GL50/B7 proteins can be used to achievedifferential activation or inhibition of target cell populations andskewing of T cell phenotypes. Such nucleic acid and polypeptidemolecules are also embraced by the invention.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding a GL50 fusion proteins. Such nucleic acid molecules,comprising at least a first nucleotide sequence encoding a GL50polypeptide, polypeptide or peptide operatively linked to a secondnucleotide sequence encoding a non-GL50 polypeptide, polypeptide orpeptide, can be prepared by standard recombinant DNA techniques.

In a preferred embodiment, a mutant GL50 polypeptide can be assayed forthe ability to: 1) costimulate (or inhibit the costimulation of, e.g.,in soluble form) the proliferation and/or effector function (e.g.,cytokine secretion (such as, for example IL-2 or IL-10) of activated Tcells; 2) bind to an anti-B7 antibody; and/or 3) bind to a GL50 ligand(e.g., to CD28, CTLA4, and/or ICOS).

In addition to the nucleic acid molecules encoding GL50 polypeptidesdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid molecule comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid molecule encoding a protein,e.g., complementary to the coding strand of a double-stranded cDNAmolecule or complementary to an mRNA sequence. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule. The antisense nucleic acid molecule can be complementary to anentire GL50 coding strand, or only to a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding GL50. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues. Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding GL50. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding GL50 disclosed herein,antisense nucleic acid molecules of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof GL50 mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of GL50mRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of GL50 mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acidmolecule of the invention can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a GL50polypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid molecule of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaseloff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave GL50 mRNA transcripts to thereby inhibittranslation of GL50 mRNA. A ribozyme having specificity for aGL50-encoding nucleic acid can be designed based upon the nucleotidesequence of a GL50 disclosed herein (e.g., SEQ ID NO:1, 3, or 5). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a GL50-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, GL50 mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Alternatively, GL50 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the GL50(e.g., the GL50 promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the GL50 gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioessays 14(12):807-15.

In yet another embodiment, the GL50 nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra; Perry-O'Keefeet al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of GL50 nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of GL50 nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., S1 nucleases (Hyrup and Nielsen(1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup and Nielsen (1996) supra; Perry-O'Keefe (1996)supra).

In another embodiment, PNAs of GL50 can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of GL50 nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup and Nielsen (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn et al. (1996) supra). Alternatively, chimericmolecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment(Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett.5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Biotechniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

III. Isolated GL50 Polypeptides and Anti-GL50 Antibodies

One aspect of the invention pertains to isolated GL50 polypeptides, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-GL50 antibodies. In oneembodiment, native GL50 polypeptides can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, GL50polypeptides are produced by recombinant DNA techniques. Alternative torecombinant expression, a GL50 polypeptide or polypeptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theGL50 polypeptide is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of GL50polypeptide in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of GL50 polypeptide having less than about 30% (bydry weight) of non-GL50 polypeptide (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-GL50 polypeptide, still more preferably less than about 10% ofnon-GL50 polypeptide, and most preferably less than about 5% non-GL50polypeptide. When the GL50 polypeptide or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of GL50 polypeptide in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of GL50 polypeptide having less than about 30% (bydry weight) of chemical precursors or non-GL50 chemicals, morepreferably less than about 20% chemical precursors or non-GL50chemicals, still more preferably less than about 10% chemical precursorsor non-GL50 chemicals, and most preferably less than about 5% chemicalprecursors or non-GL50 chemicals.

Another aspect of the invention pertains to isolated GL50 polypeptides.Preferably, the GL50 polypeptides comprise the amino acid sequenceencoded by SEQ ID NO:1, 3, or 5. In another preferred embodiment, theprotein comprises the amino acid sequence of SEQ ID NO:2, 4, or 6. Inother embodiments, the protein has at least 50%, at least 60% amino acididentity, more preferably 70% amino acid identity, more preferably 80%,and even more preferably, 90% or 95% amino acid identity with the aminoacid sequence shown in SEQ ID NO:2, 4, or 6.

In other embodiments, the invention provides isolated portions of a GL50polypeptide. GL50 polypeptides comprising a GL50 polypeptide domain.Exemplary GL50 polypeptide domains are shown in FIG. 12 and include,IgV-like, IgC-like, transmembrane, and cytoplasmic domains.

The invention further pertains to soluble forms of GL50 polypeptides.Such forms can be naturally occurring or can be engineered and cancomprise, e.g., an extracellular domain of a GL50 polypeptide. In oneembodiment, the extracellular domain of a GL50 polypeptide comprises theIgV and IgC domains after cleavage of the signal sequence, but not thetransmembrane and cytoplasmic domains of a GL50 polypeptide (e.g.,corresponding to the amino acid sequence from about amino acid 47-279 ofSEQ ID NO:2 or about amino acid 22-258 of SEQ ID NO:6).

Biologically active portions of a GL50 polypeptide include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the GL50 polypeptide, which include lessamino acids than the full length GL50 polypeptides, and exhibit at leastone activity of a GL50 polypeptide. Typically, biologically activeportions comprise a domain or motif with at least one activity of theGL50 polypeptide. A biologically active portion of a GL50 polypeptidecan be a polypeptide which is, for example, at least 10, 25, 50, 100,150, 200 or more amino acids in length.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment). In apreferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The residues at corresponding positions are then compared andwhen a position in one sequence is occupied by the same residue as thecorresponding position in the other sequence, then the molecules areidentical at that position. The percent identity between two sequences,therefore, is a function of the number of identical positions shared bytwo sequences (i.e., % identity=# of identical positions/total # ofpositions×100). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which are introduced for optimal alignment of the two sequences. As usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. A non-limiting example of a mathematical algorithm utilizedfor comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873. Such an algorithm isincorporated into the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searchescan be performed with the NBLAST program score=100, wordlength=12 toobtain nucleotide sequences homologous to the nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Research 25(17):3389.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofan algorithm utilized for the comparison of sequences is the algorithmof Myers and Miller, CABIOS (1989). Such an algorithm is incorporatedinto the ALIGN program (version 2.0 or 2.0U) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

As another example, the alignment program in Geneworks program (byOxford Molecular; e.g., version 2.5.1) can be used with the parametersset as follows: gap creation 16, extension penalty=4, scoringmatrix=fastadna.cmp, and a constant PAM factor.

Another non-limiting example of a mathematical algorithm utilized forthe alignment of protein sequences is the Lipman-Pearson algorithm(Lipman and Pearson (1985) Science 227:1435). When using theLipman-Pearson algorithm, a PAM250 weight residue table, a gap lengthpenalty of 12, a gap penalty of 4, and a Kutple of 2 can be used. Apreferred, non-limiting example of a mathematical algorithm utilized forthe alignment of nucleic acid sequences is the Wilbur-Lipman algorithm(Wilbur and Lipman (1983) Proc. Natl. Acad. Sci. USA 80:726). When usingthe Wilbur-Lipman algorithm, a window of 20, gap penalty of 3, Ktuple of3 can be used. Both the Lipman-Pearson algorithm and the Wilbur-Lipmanalgorithm are incorporated, for example, into the MegAlign program(e.g., version 3.1.7) which is part of the DNASTAR sequence analysissoftware package.

Additional algorithms for sequence analysis are known in the art, andinclude ADVANCE and ADAM., described in Torelli and Robotti (1994)Comput. Appl. Biosci. 10:3; and FASTA, described in Pearson and Lipman(1988) Proc. Natl. Acad. Sci. USA 85:2444.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the GAP program in the GCG softwarepackage, using either a Blosum 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package, using a NWSgapdna. CMP matrix and a gap weightof 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.

Protein alignments can also be made using the Geneworks global proteinalignment program (e.g., version 2.5.1) with the cost to open gap set at5, the cost to lengthen gap set at 5, the minimum diagonal length set at4, the maximum diagonal offset set at 130, the consensus cutoff set at50% and utilizing the Parn 250 matrix.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to GL50 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to GL50polypeptide molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. For example,the nucleotide sequences of the invention can be analyzed using thedefault Blastn matrix 1-3 with gap penalties set at: existence 11 andextension 1. The amino acid sequences of the invention can be analyzedusing the default settings: the Blosum62 matrix with gap penalties setat existence 11 and extension 1. See http://www.ncbi.nlm.nih.gov.

The presence of divergent carboxyl regions on RACE clones illustrated bysequence alignments suggest that alternate signaling functions may beperformed by these distinct molecules by the additional tyrosines in theintracellular domain of these molecules. To date, only sporadic studieshave been performed to determine whether intracellular signaling foreither B7-1 or B7-2. On the basis of the presence of cytoplasmic domaintyrosines on GL50 sequences, one can predict that such signaling eventsexist. Inspection of the cytoplasmic domains of mouse and human B7-1 andB7-2 show negligible similarity and it has also been suggested that theB7 cytoplasmic domain may be completely dispensable, based on thereported ability of B7 molecules to function in gpi-anchored constructscompletely lacking cytoplasmic sequences. Accordingly, in oneembodiment, tyrosine residues in the intracellular domain of a GL50tyrosine molecule can be altered to modulate intracellular signally viaa GL50 polypeptide.

The invention also provides GL50 chimeric or fusion proteins. As usedherein, a GL50 “chimeric protein” or “fusion protein” comprises a GL50polypeptide operatively linked to a non-GL50 polypeptide. An “GL50polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to GL50 polypeptide, whereas a “non-GL50 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the GL50 polypeptide,e.g., a protein which is different from the GL50 polypeptide and whichis derived from the same or a different organism. Within a GL50 fusionprotein the GL50 polypeptide can correspond to all or a portion of aGL50 polypeptide. In a preferred embodiment, a GL50 fusion proteincomprises at least one biologically active portion of a GL50polypeptide, e.g., an extracellular domain of a GL50 polypeptide. Withinthe fusion protein, the term “operatively linked” is intended toindicate that the GL50 polypeptide and the non-GL50 polypeptide arefused in-frame to each other. The non-GL50 polypeptide can be fused tothe N-terminus or C-terminus of the GL50 polypeptide.

For example, in one embodiment, the fusion protein is a GST-GL50 memberfusion protein in which the GL50 member sequences are fused to theC-terminus of the GST sequences. In another embodiment, the fusionprotein is a GL50 member-HA fusion protein in which the GL50 membernucleotide sequence is inserted in a vector such as pCEP4-HA vector(Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082) such that theGL50 member sequences are fused in frame to an influenza hemagglutininepitope tag. Such fusion proteins can facilitate the purification of arecombinant GL50 member or can be used when a molecule that does notbind to an Fc receptor is desired.

A GL50 fusion protein can be produced by recombinant expression of anucleotide sequence encoding a first peptide having GL50 activity and anucleotide sequence encoding second peptide corresponding to a moietythat alters the solubility, affinity, stability or valency of the firstpeptide, for example, an immunoglobulin constant region. Preferably, thefirst peptide consists of a portion of the of a GL50 polypeptide (e.g.,a portion of amino acid residues (after cleavage of a signal sequence,e.g., corresponding to about amino acids 1-44 of SEQ ID NO:2) of thesequence shown in SEQ ID NO:2, 4, or 6 that is sufficient to costimulateactivated T cells. The second peptide can include an immunoglobulinconstant region, for example, a human Cγ1 domain or Cγ4 domain (e.g.,the hinge, CH2 and CH3 regions of human IgCγ1, or human IgCγ4, see e.g.,Capon et al. U.S. Pat. Nos. 5,116,964, 5,580,756, 5,844,095 and thelike, incorporated herein by reference).

Particularly preferred GL50 Ig fusion proteins include the extracellulardomain portion or variable region-like domain of a hGL50 coupled to animmunoglobulin constant region. The immunoglobulin constant region maycontain genetic modifications which reduce or eliminate effectoractivity inherent in the immunoglobulin structure. For example, DNAencoding the extracellular portion of a GL50 polypeptide can be joinedto DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 and/orIgCγ4 modified by site directed mutagenesis, e.g., as taught in WO97/28267.

The nucleotide and amino acid sequences of exemplary soluble GL50 andICOS constructs are presented in FIGS. 26-29. FIG. 26 sets forthexemplary human ICOS fusion protein nucleic acid and amino acidsequence, FIG. 27 sets forth an exemplary murine ICOS fusion proteinnucleic acid and amino acid sequence, FIG. 28 sets forth an exemplaryhuman GL50 fusion protein nucleic acid and amino acid sequence, and FIG.29 sets forth an exemplary murine GL50 fusion protein nucleic acid andamino acid sequence.

A resulting GL50-Ig fusion protein may have altered solubility, bindingaffinity, stability and/or valency (i.e., the number of binding sitesavailable per molecule) and may increase the efficiency of proteinpurification. Fusion proteins and peptides produced by recombinanttechniques may be secreted and isolated from a mixture of cells andmedium containing the protein or peptide. Alternatively, the protein orpeptide may be retained cytoplasmically and the cells harvested, lysedand the protein isolated. A cell culture typically includes host cells,media and other byproducts. Suitable media for cell culture are wellknown in the art. Protein and peptides can be isolated from cell culturemedia, host cells, or both using techniques known in the art forpurifying proteins and peptides. Techniques for transfecting host cellsand purifying proteins and peptides are known in the art.

Preferably, a GL50 fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, Ausubel et al. eds.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide or an HA epitope tag). A GL50 encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the GL50 polypeptide.

In another embodiment, the fusion protein is a GL50 polypeptidecontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofGL50 can be increased through use of a heterologous signal sequence.

The GL50 fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. Useof GL50 fusion proteins may be useful therapeutically for the treatmentof immunological disorders, e.g., autoimmune diseases or in the case oftransplantation. Moreover, the GL50-fusion proteins of the invention canbe used as immunogens to produce anti-GL50 antibodies in a subject, topurify GL50 ligands and in screening assays to identify molecules whichinhibit the interaction of GL50 with a GL50 ligand.

The present invention also pertains to variants of the GL50 polypeptideswhich function as either GL50 agonists (mimetics) or as GL50antagonists. Variants of the GL50 polypeptides can be generated bymutagenesis, e.g., discrete point mutation or truncation of a GL50polypeptide. An agonist of the GL50 polypeptides can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of a GL50 polypeptide. An antagonist of a GL50polypeptide can inhibit one or more of the activities of the naturallyoccurring form of the GL50 polypeptide by, for example, competitivelymodulating a cellular activity of a GL50 polypeptide. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the GL50polypeptide.

In one embodiment, variants of a GL50 polypeptide which function aseither GL50 agonists (mimetics) or as GL50 antagonists can be identifiedby screening combinatorial libraries of mutants, e.g., truncationmutants, of a GL50 polypeptide for GL50 polypeptide agonist orantagonist activity. In one embodiment, a variegated library of GL50variants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof GL50 variants can be produced by, for example, enzymatically ligatinga mixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential GL50 sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of GL50 sequences therein.There are a variety of methods which can be used to produce libraries ofpotential GL50 variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential GL50 sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acids Res. 11:477.

In addition, libraries of fragments of a GL50 polypeptide codingsequence can be used to generate a variegated population of GL50fragments for screening and subsequent selection of variants of a GL50polypeptide. In one embodiment, a library of coding sequence fragmentscan be generated by treating a double stranded PCR fragment of a GL50coding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of theGL50 polypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of GL50 polypeptides. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify GL50 variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delagrave et al. (1993) Protein Eng. 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated GL50 library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily synthesizes andsecretes GL50. The transfected cells are then cultured such that GL50and a particular mutant GL50 are secreted and the effect of expressionof the mutant on GL50 activity in cell supernatants can be detected,e.g., by any of a number of enzymatic assays. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of GL50 activity, and the individual clones furthercharacterized.

In addition to GL50 polypeptides consisting only of naturally-occurringamino acids, GL50 peptidomimetics are also provided. Peptide analogs arecommonly used in the pharmaceutical industry as non-peptide drugs withproperties analogous to those of the template peptide. These types ofnon-peptide compound are termed “peptide mimetics” or “peptidomimetics”(Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985)TINS p. 392; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated herein by reference) and are usually developed with the aidof computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides can be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biological or pharmacological activity),such as human GL50, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH2NH—,—CH2S—, —CH2—CH2-, —CH═CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and—CH2SO—, by methods known in the art and further described in thefollowing references: Spatola, A. F. in “Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins” Weinstein, B., ed., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, “Peptide Backbone Modifications” (general review); Morley, J.S. (1980) Trends Pharm. Sci. pp. 463-468 (general review); Hudson, D. etal. (1979) Int. J. Pept. Prot. Res. 14:177-185 (—CH2NH—, CH2CH2-);Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249 (—CH2-S); Hann, M.M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314 (—CH—CH—, cis andtrans); Almquist, R. G. et al. (190) J. Med. Chem. 23:1392-1398(—COCH2-); Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533(—COCH2-); Szelke, M. et al. European Appln. EP 45665 (1982) CA:97:39405 (1982)(—CH(OH)CH2-); Holladay, M. W. et al. (1983) TetrahedronLett. (1983) 24:4401-4404 (—C(OH)CH2-); and Hruby, V. J. (1982) LifeSci. (1982) 31:189-199 (—CH2-S—); each of which is incorporated hereinby reference. A particularly preferred non-peptide linkage is —CH2NH—.Such peptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics usually involves covalent attachmentof one or more labels, directly or through a spacer (e.g., an amidegroup), to non-interfering position(s) on the peptidomimetic that arepredicted by quantitative structure-activity data and/or molecularmodeling. Such non-interfering positions generally are positions that donot form direct contacts with the macromolecules(s) to which thepeptidomimetic binds to produce the therapeutic effect. Derivitization(e.g., labeling) of peptidomimetics should not substantially interferewith the desired biological or pharmacological activity of thepeptidomimetic.

Systematic substitution of one or more amino acids of a GL50 amino acidsequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) can be used to generate more stable peptides. In addition,constrained peptides comprising a GL50 amino acid sequence or asubstantially identical sequence variation can be generated by methodsknown in the art (Rizo and Gierasch (1992) Annu. Rev. Biochem. 61:387,incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

The amino acid sequences of GL50 polypeptides identified herein willenable those of skill in the art to produce polypeptides correspondingto GL50 peptide sequences and sequence variants thereof. Suchpolypeptides can be produced in prokaryotic or eukaryotic host cells byexpression of polynucleotides encoding a GL50 peptide sequence,frequently as part of a larger polypeptide. Alternatively, such peptidescan be synthesized by chemical methods. Methods for expression ofheterologous proteins in recombinant hosts, chemical synthesis ofpolypeptides, and in vitro translation are well known in the art and aredescribed further in Maniatis et al. Molecular Cloning: A LaboratoryManual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel,Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques(1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969)J. Am. Chem. Soc. 91:501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem.11: 255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986)Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; andOfford, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which areincorporated herein by reference).

Peptides can be produced, typically by direct chemical synthesis, andused e.g., as agonists or antagonists of a GL50/GL50 ligand interaction.Peptides can be produced as modified peptides, with nonpeptide moietiesattached by covalent linkage to the N-terminus and/or C-terminus. Incertain preferred embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. The most commonmodifications of the terminal amino and carboxyl groups are acetylationand amidation, respectively. Amino-terminal modifications such asacylation (e.g., acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments of the invention. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others. Peptides can be usedtherapeutically to treat disease, e.g., by altering costimulation in apatient.

An isolated GL50 polypeptide, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind GL50 usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length GL50 polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of GL50 for use asimmunogens. The antigenic peptide of GL50 comprises at least 8 aminoacid residues and encompasses an epitope of GL50 such that an antibodyraised against the peptide forms a specific immune complex with GL50.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Alternatively, an antigenic peptide fragment of a GL50 polypeptide canbe used as the immunogen. An antigenic peptide fragment of a GL50polypeptide typically comprises at least 8 amino acid residues of theamino acid sequence shown in SEQ ID NO:2, 4, or 6 and encompasses anepitope of a GL50 polypeptide such that an antibody raised against thepeptide forms an immune complex with a GL50 molecule. Preferred epitopesencompassed by the antigenic peptide are regions of GL50 that arelocated on the surface of the protein, e.g., hydrophilic regions. In oneembodiment, an antibody binds substantially specifically to a GL50molecule. In another embodiment, an antibody binds specifically to aGL50 polypeptide.

Preferably, the antigenic peptide comprises at least about 10 amino acidresidues, more preferably at least about 15 amino acid residues, evenmore preferably at least 20 about amino acid residues, and mostpreferably at least about 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of a GL50 polypeptidethat are located on the surface of the protein, e.g., hydrophilicregions, and that are unique to a GL50 polypeptide. In one embodimentsuch epitopes can be specific for a GL50 polypeptides from one species,such as mouse or human (i.e., an antigenic peptide that spans a regionof a GL50 polypeptide that is not conserved across species is used asimmunogen; such non conserved residues can be determined using analignment such as that provided herein). A standard hydrophobicityanalysis of the GL50 polypeptide can be performed to identifyhydrophilic regions.

A GL50 immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinantly expressed GL50 polypeptide or a chemicallysynthesized GL50 peptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic GL50 preparation induces a polyclonal anti-GL50 antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-GL50antibodies. Polyclonal anti-GL50 antibodies can be prepared as describedabove by immunizing a suitable subject with a GL50 immunogen. Theanti-GL50 antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized a GL50 polypeptide. If desired, theantibody molecules directed against a GL50 polypeptide can be isolatedfrom the mammal (e.g., from the blood) and further purified by wellknown techniques, such as protein A chromatography to obtain the IgGfraction. At an appropriate time after immunization, e.g., when theanti-GL50 antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497 (see also,Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) J. Biol.Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the morerecent human B cell hybridoma technique (Kozbor et al. (1983) Immunol.Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triomatechniques. The technology for producing monoclonal antibody hybridomasis well known (see generally Kenneth, R. H. in Monoclonal Antibodies: ANew Dimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387402; Gefter,M. L. et al. (1977) Somatic Cell Genet., 3:231-36). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with a GL50 immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds specifically to a GL50 polypeptide.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-GL50 monoclonal antibody (see, e.g., Galfre, G. et al. (1977)Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra;Kenneth, Monoclonal Antibodies, supra). Moreover, the ordinary skilledworker will appreciate that there are many variations of such methodswhich also would be useful. Typically, the immortal cell line (e.g., amyeloma cell line) is derived from the same mammalian species as thelymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line. Preferredimmortal cell lines are mouse myeloma cell lines that are sensitive toculture medium containing hypoxanthine, aminopterin and thymidine (“HATmedium”). Any of a number of myeloma cell lines may be used as a fusionpartner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines areavailable from the American Type Culture Collection (ATCC), Rockville,Md. Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind aGL50 molecule, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-GL50 antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a GL50 to thereby isolateimmunoglobulin library members that bind a GL50 polypeptide. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. International Publication No. WO 92/18619;Dower et al International Publication No. WO 91/17271; Winter et al.International Publication WO 92/20791; Markland et al InternationalPublication No. WO 92/15679; Breitling et al International PublicationWO 93/01288; McCafferty et al International Publication No. WO 92/01047;Garrard et al. International Publication No. WO 92/09690; Ladner et al.International Publication No. WO 90/02809; Fuchs et al. (1991)Biotechnology (NY) 9:1369-1372; Hay et al (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Biotechnology (NY) 9:1373-1377; Hoogenboom et al. (1991) Nucleic AcidsRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

Additionally, recombinant anti-GL50 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira et al. EuropeanPatent Application 184,187; Taniguchi, M. European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad.Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sunet al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al (1987)Cancer Res. 47:999-1005; Wood et al (1985) Nature 314:446-449; and Shawet al (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable geneic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. No. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

In one embodiment, an antibody for use in the instant invention is abispecific antibody. A bispecific antibody has binding sites for twodifferent antigens within a single antibody molecule. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies. Examplesof bispecific antibodies produced by a hybrid hybridoma or a trioma aredisclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have beenconstructed by chemical means (Staerz et al. (1985) Nature 314:628, andPerez et al. (1985) Nature 316:354) and hybridoma technology (Staerz andBevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan(1986) Immunol. Today 7:241). Bispecific antibodies are also describedin U.S. Pat. No. 5,959,084. Fragments of bispecific antibodies aredescribed in U.S. Pat. No. 5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. For example, bispecific agents that bind to the T cellreceptor complex, the B cell receptor complex, CD40, CD40 ligand, CD2,or CD45 (in addition ot GL50 or ICOS) can be developed.

An anti-GL50 antibody (e.g., monoclonal antibody) can be used to isolatea GL50 polypeptide by standard techniques, such as affinitychromatography or immunoprecipitation. Anti-GL50 antibodies canfacilitate the purification of natural GL50 polypeptides from cells andof recombinantly produced GL50 polypeptides expressed in host cells.Moreover, an anti-GL50 antibody can be used to detect a GL50 polypeptide(e.g., in a cellular lysate or cell supernatant). In addition,antibodies to GL50 can be used to block the interaction between GL50 anda ligand or binding partner. Detection can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Accordingly, in one embodiment, an anti-GL50 antibody of the inventionis labeled with a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

Yet another aspect of the invention pertains to anti-GL50 antibodiesthat are obtainable by a process comprising:

(a) immunizing an animal with an immunogenic GL50 polypeptide, or animmunogenic portion thereof unique to a GL50 polypeptide; and

(b) isolating from the animal antibodies that specifically bind to aGL50 polypeptide.

IV. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a GL50 familyprotein (or a portion thereof). As used herein, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., GL50 familyproteins, mutant forms of GL50 polypeptides or portions thereof, fusionproteins, and the like).

In one embodiment of the invention, vectors comprising only atransmembrane or intracellular domain of a GL50 molecule can beengineered. Such constructs can be used to modulate intracellularsignaling via GL50 molecules, e.g., and act as dominant negativemutants.

The recombinant expression vectors of the invention can be designed forexpression of GL50 polypeptides in prokaryotic or eukaryotic cells. Forexample, GL50 polypeptides can be expressed in bacterial cells such asE. coli, insect cells (using baculovirus expression vectors) yeast cellsor mammalian cells. Suitable host cells are discussed further in Goeddel(1990) supra. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized therapeutically, in GL50activity assays, (e.g., direct assays or competitive assays described indetail below), or to generate antibodies specific for GL50 polypeptides,for example.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression in E. coliis to express the polypeptide in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant polypeptide(Gottesman, S. (1990) Methods Enzymol. 185:119-128). Another strategy isto alter the nucleic acid sequence of the nucleic acid to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in E. coli (Wada et al. (1992)Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the GL50 expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(Invitrogen Corp, San Diego, Calif.).

Alternatively, a GL50 polypeptide can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of polypeptides in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow, V. A., and Summers, M. D. (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid molecule of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pMex-NeoI, pCDM8 (Seed,B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187-195). When used in mammalian cells, the expression vector'scontrol functions are often provided by viral regulatory elements. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. For other suitable expressionsystems for both prokaryotic and eukaryotic cells see chapters 16 and 17of Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2nd ed,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Moreover, inducible regulatory systems for use in mammalian cells areknown in the art, for example systems in which gene expression isregulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et at (1991)in Heat Shock Response, Nouer, L., ed. CRC, Boca Raton, Fla.,pp167-220), hormones (see e.g, Lee et al (1981) Nature 294:228-232;Hynes et al (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock et al(1987) Nature 329:734-736; Israel and Kaufman (1989) Nucleic Acids Res.17:2589-2604; and PCT Publication No. WO 93/23431), FK506-relatedmolecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCTPublication No. WO 94/29442; and PCT Publication No. WO 96/01313).Accordingly, in another embodiment, the invention provides a recombinantexpression vector in which a GL50 DNA is operatively linked to aninducible eukaryotic promoter, thereby allowing for inducible expressionof a GL50 polypeptide in eukaryotic cells.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to GL50 mRNA. Regulatory sequences operatively linkedto a nucleic acid molecule cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aGL50 polypeptide can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a GL50 polypeptide or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a GL50polypeptide. Accordingly, the invention further provides methods forproducing a GL50 polypeptide using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding a GL50polypeptide has been introduced) in a suitable medium such that a GL50polypeptide is produced. In another embodiment, the method furthercomprises isolating a GL50 polypeptide from the medium or the host cell.

Certain host cells of the invention can also be used to producenon-human transgenic animals. For example, in one embodiment, a hostcell of the invention is a fertilized oocyte or an embryonic stem cellinto which GL50-coding sequences have been introduced. Such host cellscan then be used to create non-human transgenic animals in whichexogenous GL50 sequences have been introduced into their genome orhomologous recombinant animals in which endogenous GL50 sequences havebeen altered. Such animals are useful for studying the function and/oractivity of a GL50 polypeptide and for identifying and/or evaluatingmodulators of GL50 activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa transgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous GL50 gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aGL50-encoding nucleic acid into the male pronucleus of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The mGL50-1sequence of SEQ ID NO:1, 3, or 5 can be introduced as a transgene intothe genome of a non-human animal. Alternatively, a nonhuman homologue ofa hGL50 gene, such as a mouse or rat GL50 gene, can be used as atransgene. Alternatively, a GL50 gene homologue, such as another GL50family member, can be isolated based on hybridization to the GL50 familycDNA sequences of SEQ ID NO:1, 3, or 5 (described further in subsectionI above) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to a GL50 transgene to direct expression of aGL50 polypeptide to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of a GL50 transgene in its genomeand/or expression of GL50 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a GL50 polypeptide can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a GL50 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the GL50 gene. The GL50 gene can be a human gene(e.g., the SEQ ID NO:1, 3, or 5), but more preferably, is a non-humanhomologue of a hGL50 gene (e.g., a cDNA isolated by stringenthybridization with the nucleotide sequence of SEQ ID NO:1, 3, or 5). Forexample, a mouse GL50 gene can be used to construct a homologousrecombination vector suitable for altering an endogenous GL50 gene inthe mouse genome. In a preferred embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous GL50 gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousGL50 gene is mutated or otherwise altered but still encodes a functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous GL50 polypeptide). In thehomologous recombination vector, the altered portion of the GL50 gene isflanked at its 5′ and 3′ ends by additional nucleic acid sequence of theGL50 gene to allow for homologous recombination to occur between theexogenous GL50 gene carried by the vector and an endogenous GL50 gene inan embryonic stem cell. The additional flanking GL50 nucleic acidsequence is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see e.g.,Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a descriptionof homologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced GL50 gene has homologously recombined with the endogenousGL50 gene are selected (see, e.g., Li, E. et al. (1992) Cell 69:915).The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opinion in Biotechnology2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

In addition to the foregoing, the skilled artisan will appreciate thatother approaches known in the art for homologous recombination can beapplied to the instant invention. Enzyme-assisted site-specificintegration systems are known in the art and can be applied to integratea DNA molecule at a predetermined location in a second target DNAmolecule. Examples of such enzyme-assisted integration systems includethe Cre recombinase-lox target system (e.g., as described in Baubonis,W. and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S.and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and theFLP recombinase-FRT target system (e.g., as described in Dang, D. T. andPerrimon, N. (1992) Dev. Genet. 13:367-375; and Fiering, S. et al.(1993) Proc. Natl. Acad. Sci. USA 90:8469-8473). Tetracycline-regulatedinducible homologous recombination systems, such as described in PCTPublication No. WO 94/29442 and PCT Publication No. WO 96/01313, alsocan be used.

For example, in another embodiment, transgenic non-humans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinasesystem is used to regulate expression of the transgene, animalscontaining transgenes encoding both the Cre recombinase and a selectedprotein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

V. Pharmaceutical Compositions

GL50 modulators (“active compounds”) of the invention (e.g., GL50inhibitory or stimulatory agents, including GL50 nucleic acid molecules,polypeptides, antibodies, or compounds identified as modulators of aGL50 activity) can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, polypeptide, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a GL50 polypeptide, nucleic acid molecule, or anti-GL50antibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The nucleic acid molecules, polypeptides, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: a) methods of treatment, e.g., up- or down-modulating theimmune response; b) screening assays; c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenetics). The isolated nucleic acid molecules of the inventioncan be used, for example, to express GL50 polypeptide (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect GL50 mRNA (e.g., in a biological sample) or agenetic alteration in a GL50 gene, and to modulate GL50 activity, asdescribed further below. The GL50 polypeptides can be used to treatdisorders characterized by insufficient or excessive production of GL50inhibitors. In addition, the GL50 polypeptides can be used to screen fornaturally occurring GL50 ligands, to screen for drugs or compounds whichmodulate GL50 activity, as well as to treat disorders characterized byinsufficient or excessive production of GL50 polypeptide or productionof GL50 polypeptide forms which have decreased or aberrant activitycompared to GL50 wild type polypeptide. Moreover, the anti-GL50antibodies of the invention can be used to detect and isolate GL50polypeptides, regulate the bioavailability of GL50 polypeptides, andmodulate GL50 activity e.g., modulate immune responses.

A. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant GL50 expression oractivity or a disorder that would benefit from modulation of GL50activity.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant GL50expression or activity, by administering to the subject a GL50polypeptide or an agent which modulates GL50 polypeptide expression orat least one GL50 activity. Subjects at risk for a disease which iscaused or contributed to by aberrant GL50 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofGL50 aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of GL50aberrancy or condition, for example, a GL50 polypeptide, GL50 agonist orGL50 antagonist agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating GL50expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a GL50 polypeptide or agent that modulates one ormore of the activities of GL50 polypeptide associated with the cell. Anagent that modulates GL50 polypeptide activity can be an agent asdescribed herein, such as a nucleic acid or a polypeptide, anaturally-occurring target molecule of a GL50 polypeptide (e.g., a GL50ligand), a GL50 antibody, a GL50 agonist or antagonist, a peptidomimeticof a GL50 agonist or antagonist, or other small molecule. In oneembodiment, the agent stimulates one or more GL50 activities. Examplesof such stimulatory agents include agents that stimulate the interactionof GL50 with a stimulatory receptor or inhibit the interaction of GL50with an inhibitory receptor, e.g., active GL50 polypeptide, certainsoluble forms of GL50 molecules, and a nucleic acid molecule encodingGL50 polypeptide that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more GL50 activities. Examples ofsuch inhibitory agents include agents that diminish the interaction ofGL50 and a costimulatory receptor or promote the interaction betweenGL50 and an inhibitory receptor, e.g., antisense GL50 nucleic acidmolecules, anti-GL50 antibodies, and GL50 inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder that would benefit frommodulation of a GL50 polypeptide, e.g., a disorder which would benefitfrom up- or down-modulation of the immune response, or which ischaracterized by aberrant expression or activity of a GL50 polypeptideor nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) GL50 expression or activity. In anotherembodiment, the method involves administering a GL50 polypeptide ornucleic acid molecule as therapy to compensate for reduced or aberrantGL50 expression or activity.

Stimulation of GL50 activity is desirable in situations in which GL50 isabnormally downregulated and/or in which increased GL50 activity islikely to have a beneficial effect. Likewise, inhibition of GL50activity is desirable in situations in which GL50 is abnormallyupregulated and/or in which decreased GL50 activity is likely to have abeneficial effect.

3. Downregulation of Immune Responses

It is possible to downregulate the function of a GL50 polypeptide, andthereby downregulate immune responses, in a number of ways.Downregulation may be in the form of inhibiting or blocking an immuneresponse already in progress or may involve preventing the induction ofan immune response. The functions of activated T cells may be inhibited,e.g., by suppressing T cell responses or by inducing specific tolerancein T cells, or by leading to the production of cytokines that dampen theimmune response. Immunosuppression of T cell responses is generally anactive, non-antigen-specific, process which leads to decreased T cellresponsiveness and may require continuous exposure of the T cells to thesuppressive agent. Tolerance, which involves inducing non-responsivenessor anergy in T cells, is distinguishable from immunosuppression in thatit is generally antigen-specific and persists after exposure to thetolerizing agent has ceased. Operationally, tolerance can bedemonstrated by the lack of a T cell response upon reexposure tospecific antigen where the reexposure occurs in the absence of thetolerizing agent.

For example, GL50 polypeptides, (including nonactivating forms of a GL50polypeptide) or anti-GL50 antibodies that result in the failure todeliver a costimulatory signal to T cells that have received a primaryactivation signal, can be used to block GL50 the interaction betweenGL50 and its ligand(s) on T cells and thereby provide a specific meansby which to cause immunosuppression and/or induce tolerance in asubject. Such blocking or inhibitory forms of GL50 polypeptides andfusion proteins and blocking antibodies can be identified by theirability to inhibit T cell proliferation and/or cytokine production whenadded to an in vitro costimulation assay as described herein and knownin the art. In contrast to the inhibitory forms of a GL50 polypeptide,activating forms (such as an intact cell surface GL50 polypeptide andcertain soluble forms of GL50) preferably transmit a costimulatorysignal to the T cells, resulting in an increased secretion of cytokines(e.g., IL-10) when compared to activated T cells that have not receiveda costimulatory signal.

In one embodiment, fusion proteins comprising a GL50 first peptide fusedto a second peptide having an activity of another B lymphocyte antigen(e.g., B7-1 or B7-2) can be used to modify T cell mediated immuneresponses. Alternatively, two separate peptides having an activity of Blymphocyte antigens, (for example, a GL50 polypeptide plus a B7-2 and/orB7-1 polypeptide), or a combination of blocking antibodies (e.g.,antibodies against a GL50 polypeptide with anti-B7-2 and/or anti-B7-1monoclonal antibodies) can be combined as a single composition oradministered separately (simultaneously or sequentially), to upregulateor downregulate T cell mediated immune responses in a subject.Furthermore, a therapeutically active amount of one or more peptideshaving a GL50 polypeptide activity, with B7-1 and/or B7-2 activity canbe used in conjunction with other immunomodulating reagents to influenceimmune responses. Examples of other immunomodulating reagents includeblocking antibodies, (e.g., against CD28, CTLA4, and/or ICOS, or againstother T cell markers, or against cytokines), fusion proteins (e.g.,CTLA4Ig), or immunosuppressive drugs, (e.g., rapamycin, cyclosporine Aor FK506).

The peptides produced from the nucleic acid molecules of the presentinvention may also be useful in the construction of therapeutic agentswhich block T cell function by destruction of the T cell. For example,as described, soluble, secreted forms of a GL50 polypeptide orantibodies that bind to a ligand on a T cell can be used. Such secretedforms can be constructed by standard genetic engineering techniques. Bylinking a soluble form of a GL50 polypeptide or antibody to a toxin suchas ricin, an agent capable of preventing T cell activation can be made.Infusion of one or a combination of immunotoxins, (e.g., GL50-ricin withB7-2-ricin and/or B7-1-ricin), into a patient may result in the death ofT cells, particularly of activated T cells that express higher amountsof CD28, CTLA4, and/or ICOS or GL50.

Another method of preventing the function of a GL50 polypeptide isthrough the use of an antisense or triplex oligonucleotide. For example,an oligonucleotide complementary to the area around a GL50 polypeptidetranslation initiation site, can be synthesized. One or more antisenseoligonucleotides can be added to cell media, typically at 200 μg/ml, oradministered to a patient to prevent the synthesis of a GL50polypeptide. The antisense oligonucleotide is taken up by cells andhybridizes to a GL50 mRNA to prevent translation. Alternatively, anoligonucleotide which binds double-stranded DNA to form a triplexconstruct to prevent DNA unwinding and transcription can be used. As aresult of either, synthesis of a GL50 polypeptide is blocked.

Downregulating or preventing one or more GL50 polypeptide functions,e.g., preventing high level lymphokine synthesis by activated T cells,will be useful in situations of tissue, skin and organ transplantationand in graft-versus-host disease (GVHD). For example, blockage of T cellfunction should result in reduced tissue destruction in tissuetransplantation. Typically, in tissue transplants, rejection of thetransplant is initiated through its recognition as foreign by T cells,followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a GL50 polypeptide alone or inconjunction with a monomeric form of a different B7 peptide (e.g., B7-1,B7-2) or blocking antibody), prior to transplantation can lead to thebinding of the molecule to the natural ligand(s) on the immune cellswithout transmitting the corresponding costimulatory signal. Blocking Blymphocyte antigen function in this manner prevents cytokine synthesisby immune cells, such as T cells and, thus, acts as animmunosuppressant. Moreover, the lack of costimulation may also besufficient to anergize the T cells, thereby inducing tolerance in asubject. Induction of long-term tolerance by B lymphocyteantigen-blocking reagents may avoid the necessity of repeatedadministration of these blocking reagents. To achieve sufficientimmunosuppression or tolerance in a subject, it may also be necessary toblock the function of a combination of B lymphocyte antigens. Forexample, it may be desirable to block the function of B7-1 and GL50,B7-2 and GL50, or B7-1 and B7-2 and a GL50 polypetide, by administeringa soluble form of a combination of peptides having an activity of eachof these antigens or blocking antibodies against these antigens(separately or together in a single composition) prior totransplantation. Alternatively, inhibitory forms of GL50 polypeptidescan be used with other suppressive agents such as blocking antibodiesagainst other T cell markers or against cytokines, other fusionproteins, e.g., CTLA4Ig, or immunosuppressive drugs.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Because B7 polypeptides displayamino acid conservation across species, it is likely that other GL50antigens can function across species, thereby allowing use of reagentscomposed of human proteins in animal systems. Examples of appropriatesystems which can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science, 257: 789-792 (1992)and Turka et al., Proc. Natl. Acad. Sci. USA, 89: 11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking function of a GL50 polypeptide in vivo on thedevelopment of that disease.

Blocking a GL50 polypeptide function, e.g., by use of a peptide having aGL50 polypeptide activity alone or in combination with a peptide havingB7-1 activity and/or a peptide having B7-2 activity, may also betherapeutically useful for treating autoimmune diseases. Many autoimmunedisorders are the result of inappropriate activation of T cells that arereactive against self tissue and which promote the production ofcytokines and autoantibodies involved in the pathology of the diseases.Preventing the activation of autoreactive T cells may reduce oreliminate disease symptoms. Administration of reagents which blockcostimulation of T cells by disrupting receptor:ligand interactions of Blymphocyte antigens can be used to inhibit T cell activation and preventproduction of autoantibodies or T cell-derived cytokines which may beinvolved in the disease process. Additionally, blocking reagents mayinduce antigen-specific tolerance of autoreactive T cells which couldlead to long-term relief from the disease. The efficacy of blockingreagents in preventing or alleviating autoimmune disorders can bedetermined using a number of well-characterized animal models of humanautoimmune diseases. Examples include murine experimental autoimmuneencephalitis, systemic lupus erythematosus in MRL/lpr/lpr mice or NZBhybrid mice, murine autoimmune collagen arthritis, diabetes mellitus inNOD mice and BB rats, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856).

The IgE antibody response in atopic allergy is highly T cell dependentand, thus, inhibition of B lymphocyte antigen induced T cell activationmay be useful therapeutically in the treatment of allergy and allergicreactions. An inhibitory form of a GL50 polypeptide, such as a peptidehaving a GL50 polypeptide activity alone or in combination with anotherB lymphocyte antigen, such as B7-1 or B7-2, can be administered to anallergic subject to inhibit T cell mediated allergic responses in thesubject. Inhibition of GL50 costimulation of T cells may be accompaniedby exposure to allergen in conjunction with appropriate MHC molecules.Allergic reactions may be systemic or local in nature, depending on theroute of entry of the allergen and the pattern of deposition of IgE onmast cells or basophils. Thus, it may be necessary to inhibit T cellmediated allergic responses locally or systemically by properadministration of an inhibitory form of a GL50 polypeptide.

Inhibition of T cell activation through blockage of a GL50 antigenfunction may also be important therapeutically in viral infections of Tcells. For example, in the acquired immune deficiency syndrome (AIDS),viral replication is stimulated by T cell activation. Blocking a GL50function could lead to a lower level of viral replication and therebyameliorate the course of AIDS. In addition, it may also be desirable toblock the function of a combination of B lymphocyte antigens i.e., GL50with B7-2 and/or B7-1.

In one embodiment of the invention, a GL50 family member preferentiallyinduces IL-10 secretion by a T cell (Hutloff et al. (1999) Nature397:263). IL-10, while promoting the development of Th2 type responses,also leads to downmodulation of the production of certain cytokines, anda downmodulation of cell mediated immunity, e.g., by decreasingmacrophage activation (Bai et al. (1997) Clin. Immunol. Immunopathol.83:117; Koch et al. (1996) J. Exp. Med 184:741; deVries (1995) Ann. Med.27:537). Accordingly, in one embodiment of the invention, increasing theactivity of a GL50 family member can lead to downmodulation of acell-mediated immune response. Thus, in one embodiment of the inventioncell-mediated immune responses are decreased by increasing GL50activity.

4. Upregulation of Immune Responses

Upregulation of an immune response, e.g., by promoting a stimulatoryactivity of GL50 may also be useful in therapy. Upregulation of immuneresponses may be in the form of enhancing an existing immune response oreliciting an initial immune response. For example, enhancing an immuneresponse through stimulating GL50 activity may be useful in cases ofviral infection. Viral infections are cleared primarily by cytolytic Tcells. In accordance with the present invention, it is believed thatGL50 polypeptide interacting with its natural ligand(s) on T cells mayresult in an increase in the cytolytic activity of at least some Tcells. The addition of an activating form of GL50, alone, or incombination with an activating form of a different B7 family polypeptideto stimulate T cell activity through the costimulation pathway wouldthus be therapeutically useful in situations where more rapid orthorough clearance of virus would be beneficial. These would includeviral skin diseases such as Herpes or shingles, in which cases themono-valent or multi-valent soluble GL50 polypeptide or combination ofsuch peptide with a peptide having B7-1 activity and/or a peptide havingB7-2 activity is delivered topically to the skin. In addition, systemicviral diseases such as influenza, the common cold, and encephalitismight be alleviated by the administration of stimulatory forms of GL50systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing a GL50peptide (alone or in combination with a peptide having B7-1 activityand/or a peptide having B7-2 activity) or together with a stimulatoryform of a soluble GL50 peptide (alone or in combination with a peptidehaving B7-1 activity and/or a peptide having B7-2 activity) andreintroducing the in vitro activated T cells into the patient. Anothermethod of enhancing anti-viral immune responses would be to isolateinfected cells from a patient, transfect them with a nucleic acidmolecule encoding a peptide having the activity of a B lymphocyteantigen as described herein such that the cells express all or a portionof a GL50 antigen on their surface, and reintroduce the transfectedcells into the patient. The infected cells would now be capable ofdelivering a costimulatory signal to, and thereby activate, T cells invivo.

Stimulatory forms of GL50 molecules may also be used prophylactically invaccines against various pathogens. Immunity against a pathogen, e.g., avirus, could be induced by vaccinating with a viral protein along with astimulatory form of a GL50 polypeptide in an appropriate adjuvant.Alternately, an expression vector which encodes genes for both apathogenic antigen and a peptide having the activity of a GL50 antigen,e.g., a vaccinia virus expression vector engineered to express a nucleicacid molecule encoding a viral protein and a nucleic acid moleculeencoding a GL50 polypeptide as described herein, can be used forvaccination. DNA vaccines can be administered by a variety of means, forexample, by injection (e.g., intramuscular, intradermal, or thebiolistic injection of DNA-coated gold particles into the epidermis witha gene gun that uses a particle accelerator or a compressed gas toinject the particles into the skin (Haynes et al. (1996) J. Biotechnol.44:37)). Alternatively, DNA vaccines can be administered by non-invasivemeans. For example, pure or lipid-formulated DNA can be delivered to therespiratory system or targeted elsewhere, e.g., Peyers patches by oraldelivery of DNA (Schubbert (1997) Proc. Natl. Acad. Sci. USA 94:961).Attenuated microorganisms can be used for delivery to mucosal surfaces.(Sizemore et al. 1995. Science. 270:29). In one embodiment, antigen isadministered concurrently with a stimulatory form of a GL50 molecule.

In another application, upregulation or enhancement of GL50 function maybe useful in the induction of tumor immunity. In one embodiment, theGL50 molecule is cell associated. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one GL50 antigen can be administered to a subjectto overcome tumor-specific tolerance in the subject. If desired, thetumor cell can be transfected to express a combination of B7polypeptides (e.g., B7-1, B7-2, GL50). For example, tumor cells obtainedfrom a patient can be transfected ex vivo with an expression vectordirecting the expression of a GL50 polypeptide alone, or in conjunctionwith a peptide having B7-1 activity and/or B7-2 activity. Thetransfected tumor cells are returned to the patient to result inexpression of the peptides on the surface of the transfected cell.Alternatively, gene therapy techniques can be used to target a tumorcell for transfection in vivo.

The presence of the peptide having the activity of a GL50 molecule onthe surface of the tumor cell provides the necessary costimulationsignal to T cells to induce a T cell mediated immune response againstthe transfected tumor cells. In addition, tumor cells which lack MHCclass I or MHC class II molecules, or which fail to express sufficientamounts of MHC class I or MHC class II molecules, can be transfectedwith nucleic acid encoding all or a portion of (e.g., acytoplasmic-domain truncated portion) of an MHC class I a chain proteinand β₂ microglobulin protein or an MHC class II α chain protein and anMHC class II β chain protein to thereby express MHC class I or MHC classII proteins on the cell surface. Expression of the appropriate class Ior class II MHC in conjunction with a peptide having the activity of a Blymphocyte antigen (e.g., B7-1, B7-2, GL50) induces a T cell mediatedimmune response against the transfected tumor cell. Optionally, a geneencoding an antisense construct which blocks expression of an MHC classII associated protein, such as the invariant chain, can also becotransfected with a DNA encoding a GL50 polypeptide to promotepresentation of tumor associated antigens and induce tumor specificimmunity. Expression of B7-1 by B7 negative murine tumor cells has beenshown to induce T cell mediated specific immunity accompanied by tumorrejection and prolonged protection to tumor challenge in mice (Chen, L.et al. (1992) Cell 71:1093-1102; Townsend, S. E. and Allison, J. P.(1993) Science 259:368-370; Baskar, S. et al. (1993) Proc. Natl. Acad.Sci. USA 90:5687-5690). Thus, the induction of a T cell mediated immuneresponse in a human subject may be sufficient to overcome tumor-specifictolerance in the subject.

In another embodiment, an activating form of one or more GL50 peptides(e.g., expressed on a cell surface) can be administered to atumor-bearing patient to provide a costimulatory signal to T cells inorder to induce anti-tumor immunity using techniques that are known inthe art.

In a specific embodiment, T cells are obtained from a subject andcultured ex vivo to expand the population of T cells. In a furtherembodiment the T cells are then administered to a subject. T cells canbe stimulated to proliferate in vitro by, for example, providing to theT cells a primary activation signal and a costimulatory signal, as isknown in the art. Various forms of GL50 polypeptides can also be used tocostimulate proliferation of T cells. In one embodiment T cells arecultured ex vivo according to the method described in PCT ApplicationNo. WO 94/29436. The costimulatory molecule can be soluble, attached toa cell membrane or attached to a solid surface, such as a bead.

B. Identification of Cytokines Induced by GL50 Mediated Costimulation

The GL50 molecules as described herein can be used to identify cytokineswhich are produced by T cells in response to stimulation by a GL50polypeptide. T cells can be suboptimally stimulated in vitro with aprimary activation signal, such as phorbol ester, anti-CD3 antibody orpreferably antigen in association with an MHC class II molecule, andgiven a costimulatory signal by a stimulatory form of GL50 antigen, forinstance by a cell transfected with nucleic acid encoding a GL50polypeptide and expressing the peptide on its surface or by a soluble,stimulatory form of the peptide. Known cytokines released into the mediacan be identified by ELISA or by the ability of an antibody which blocksthe cytokine to inhibit T cell proliferation or proliferation of othercell types that is induced by the cytokine. An IL-4 ELISA kit isavailable from Genzyme (Cambridge Mass.), as is an IL-7 blockingantibody. Blocking antibodies against IL-9 and IL-12 are available fromGenetics Institute (Cambridge, Mass.).

An in vitro T cell costimulation assay as described above can also beused in a method for identifying novel cytokines which may be induced bycostimulation. For example, where stimulation of the CD28/CTLA4 pathwayseems to enhance IL-2 secretion, stimulation of the ICOS pathway seemsto enhance IL-10 secretion (Hutloff et al. 199. Nature 397:263). If aparticular activity induced upon costimulation, e.g., T cellproliferation, cannot be inhibited by addition of blocking antibodies toknown cytokines, the activity may result from the action of an unknowncytokine. Following costimulation, this cytokine could be purified fromthe media by conventional methods and its activity measured by itsability to induce T cell proliferation.

To identify cytokines which may prevent the induction of tolerance, anin vitro T cell costimulation assay as described above can be used. Inthis case, T cells would be given the primary activation signal andcontacted with a selected cytokine, but would not be given thecostimulatory signal. After washing and resting the T cells, the cellswould be rechallenged with both a primary activation signal and acostimulatory signal. If the T cells do not respond (e.g., proliferateor produce cytokines) they have become tolerized and the cytokine hasnot prevented the induction of tolerance. However, if the T cellsrespond, induction of tolerance has been prevented by the cytokine.Those cytokines which are capable of preventing the induction oftolerance can be targeted for blockage in vivo in conjunction withreagents which block B lymphocyte antigens as a more efficient means toinduce tolerance in transplant recipients or subjects with autoimmunediseases. For example, one could administer a GL50 blocking reagenttogether with a cytokine blocking antibody to a subject.

C. Identification of Molecules which Influence Costimulation

Another application of the peptide having the activity of a novel Blymphocyte antigen of the invention is the use of one or more of thesepeptides in screening assays to discover as yet undefined moleculeswhich are modulators of costimulatory ligand binding and/or ofintracellular signaling through T cells following costimulation. Forexample, a solid-phase binding assay using a peptide having the activityof a GL50 molecule, could be used to identify molecules to which GL50binds and/or which inhibit binding of the antigen with an appropriate Tcell ligand (e.g., CD28, CTLA4, or ICOS). In addition, an in vitro Tcell costimulation assay as described above could be used to identifymolecules which interfere with intracellular signaling through the Tcells following costimulation as determined by the ability of thesemolecules to inhibit T cell proliferation and/or cytokine production(yet which do not prevent binding of a GL50 molecule to its ligand). Forexample, the compound cyclosporine A and rapamycin inhibit T cellactivation through stimulation via the T cell receptor pathway but notvia the CD28/CTLA4 pathway. Therefore, a different intracellularsignaling pathway is involved in costimulation. Molecules whichinterfere with intracellular signaling via the CD28/CTLA4 and/or ICOSpathway may be effective as immunosuppressive agents in vivo with orwithout the use of an additional immunosuppressant such as cyclosporineA or rapamycin.

D. Identification of Molecules which Modulate Expression of a GL50Polypeptide

The antibodies produced using the proteins and peptides of the currentinvention can be used in a screening assay for molecules which modulatethe expression of GL50 polypeptide on cells. For example, moleculeswhich effect intracellular signaling which leads to induction ofexpression GL50 polypeptides e.g., in response to activation signals,can be identified by assaying expression of one or more GL50polypeptides on the cell surface. Reduced immunofluorescent staining byan anti-GL50 antibody in the presence of the molecule would indicatethat the molecule inhibits intracellular signals. Molecules whichupregulate GL50 polypeptide expression result in an increasedimmunofluorescent staining. Alternatively, the effect of a molecule onexpression of a GL50 polypeptide can be determined by detecting cellularGL50 mRNA levels using a probe of the invention. For example, a cellwhich expresses a GL50 polypeptide can be contacted with a molecule tobe tested, and an increase or decrease in GL50 mRNA levels in the celldetected by standard technique, such as Northern hybridization analysisor conventional dot blot of mRNA or total poly(A⁺)RNAs using a mGL50-1probe labeled with a detectable marker. Molecules which modulateexpression of a GL50 polypeptide may be useful therapeutically foreither upregulating or downregulating immune responses alone or inconjunction with soluble blocking or stimulating reagents. For instance,a molecule which inhibits expression of GL50 could be administeredtogether with a GL50 blocking reagent for immunosuppressive purposes.Molecules which can be tested in the above-described assays includecytokines such as IL-4, γINF, IL-10, IL-12, GM-CSF and prostagladins.

E. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to GL50 polypeptides or portions thereof, have a stimulatoryor inhibitory effect on, for example, GL50 expression or GL50 activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a GL50polypeptide or polypeptide or biologically active portion thereof, e.g.,modulate the ability of GL50 polypeptide to interact with a bindingpartner (e.g., a cognate ligand or intracellular interactor). Forexample, in one embodiment, portions of the extracellular domain of GL50can be used. In another embodiment, portions of the cytoplasmic domainof a GL50 molecule can be used. In another embodiment, portions of thetransmembrane domain of a GL50 molecule can be used.

In one embodiment, variant forms of a polypeptide comprising a GL50domain can be used in a screening assay. For example, GL50 domainscomprising an amino acid alteration (e.g., that have been mutagenizedusing, for example random or cassette mutagenesis) can be used in thesubject screening assays. Alternatively, splicing variants of GL50intracellular domains (e.g., GL50-1 intracellular domain, GL50-2cytoplasmic domain or additional exons identified upon sequencing ofchromosome 21 or identified by RACE PCR) can be to screen for compounds.Such GL50 variants can be used to identify compounds with activityagainst a range of GL50 molecules and can identify amino acid residuesessential for GL50 activity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci 87:6378-6382);(Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a GL50 target molecule (e.g., a GL50 ligandsuch as ICOS or intracellular interactor molecule) with a test compoundand determining the ability of the test compound to modulate (e.g.stimulate or inhibit) the activity of the GL50 target molecule. In oneembodiment, a GL50 target molecule is identified, e.g., in a two orthree hybrid assay. In another embodiment, a GL50 interactor molecule isidentified using standard methods for crosslinking GL50 to neighboringmolecules followed by immunoprecipitation using anti-GL50 antibodies.

In one embodiment, portions of the transmembrane and/or intracellularregions as defined by hydropathy plots or domains as defined by exonstructure can be as bait in 2-hybrid assays to determine bindingpartners to these domains. Interacting proteins can be used in assays toquantitate the degree of GL50 binding to interaction partnerspotentially for production or quality control assays. In anotherembodiment, cytoplasmic domain splice variants can be used in different2-hybrid assays to collect the entire range of protein interactors thatbind to any GL50 splice variant.

Determining the ability of the test compound to modulate the activity ofa GL50 target molecule can be accomplished, for example, by determiningthe ability of the GL50 polypeptide to bind to or interact with the GL50target molecule or its ligand. Determining the ability of the GL50polypeptide to bind to or interact with a ligand of a GL50 molecule canbe accomplished, e.g., by direct binding.

In a direct binding assay, the GL50 polypeptide could be coupled with aradioisotope or enzymatic label such that binding of the GL50polypeptide to a GL50 target molecule can be determined by detecting thelabeled GL50 polypeptide in a complex. For example, GL50 molecules,e.g., GL50 polypeptides, can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,GL50 molecules can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interaction between GL50 and its targetmolecule, without the labeling of any of the interactants. For example,a microphysiometer can be used to detect the interaction of GL50 withits target molecule without the labeling of either GL50 or the targetmolecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As usedherein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

In a preferred embodiment, determining the ability of the GL50polypeptide to bind to or interact with a GL50 binding partner can beaccomplished by determining the activity of the binding partner. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular second messenger of the target (e.g.,to phosphorylate GL50 or another substrate on tyrosine residues),detecting catalytic/enzymatic activity of the target an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a target-regulated cellular response. Forexample, determining the ability of the GL50 polypeptide to bind to orinteract with a GL50 target molecule can be accomplished, for example,by measuring the ability of a compound to downmodulate T cellcostimulation in a proliferation assay, or by interfering with theability of a GL50 polypeptide to bind to antibodies that recognize aportion of the GL50 polypeptide.

In yet another embodiment, an assay of the present invention is acell-free assay in which a GL50 polypeptide or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to bind to the GL50 polypeptide or biologically activeportion thereof is determined. Binding of the test compound to the GL50polypeptide can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the GL50polypeptide or biologically active portion thereof with a known compoundwhich binds GL50 to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a GL50 polypeptide, wherein determining the ability ofthe test compound to interact with a GL50 polypeptide comprisesdetermining the ability of the test compound to preferentially bind toGL50 polypeptide or biologically active portion thereof as compared tothe known compound.

In another embodiment, the assay is a cell-free assay in which a GL50polypeptide or biologically active portion thereof is contacted with atest compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the GL50 polypeptide orbiologically active portion thereof is determined. Determining theability of the test compound to modulate the activity of a GL50polypeptide can be accomplished, for example, by determining the abilityof the GL50 polypeptide to bind to a GL50 target molecule or ligand byone of the methods described above for determining direct binding.Determining the ability of the GL50 polypeptide to bind to a GL50 targetmolecule can also be accomplished using a technology such as real-timeBiomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky,C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699-705. As used herein, “BIA” is a technology forstudying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a GL50 polypeptide can beaccomplished by determining the ability of the GL50 polypeptide tofurther modulate the activity of a GL50 target molecule (e.g., a GL50mediated signal transduction pathway component). For example, theactivity of the effector molecule on an appropriate target can bedetermined, or the binding of the effector to an appropriate target canbe determined as previously described.

In yet another embodiment, the cell-free assay involves contacting aGL50 polypeptide or biologically active portion thereof with a knowncompound which binds the GL50 polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the GL50 polypeptide,wherein determining the ability of the test compound to interact withthe GL50 polypeptide comprises determining the ability of the GL50polypeptide to preferentially bind to or modulate the activity of a GL50target molecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., GL50polypeptides or biologically active portions thereof, or receptors towhich GL50 binds). In the case of cell-free assays in which amembrane-bound form a protein is used (e.g., a cell surface GL50receptor) it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the protein is maintained in solution.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In one embodiment of the above assay methods of the present invention,it may be desirable to immobilize either GL50 or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Binding of a test compound to a GL50 polypeptide, or interaction of aGL50 polypeptide with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-5-transferase/GL50 fusion proteins orglutathione-5-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or GL50 polypeptide, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of GL50binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a GL50polypeptide or a GL50 target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated GL50 polypeptide ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with GL50 polypeptide or target molecules but whichdo not interfere with binding of the GL50 polypeptide to its targetmolecule can be derivatized to the wells of the plate, and unboundtarget or GL50 polypeptide trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the GL50 polypeptide or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the GL50 polypeptide or targetmolecule.

In another embodiment, modulators of GL50 expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of GL50 mRNA or protein in the cell is determined. The levelof expression of GL50 mRNA or protein in the presence of the candidatecompound is compared to the level of expression of GL50 mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of GL50 expression based on thiscomparison. For example, when expression of GL50 mRNA or protein isgreater (e.g., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as a stimulator of GL50 mRNA or protein expression.Alternatively, when expression of GL50 mRNA or protein is less (e.g.,statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of GL50 mRNA or protein expression. The level of GL50 mRNA orprotein expression in the cells can be determined by methods describedherein for detecting GL50 mRNA or protein.

In yet another aspect of the invention, the GL50 polypeptides, e.g.,soluble or membrane bound molecules or portions thereof (e.g.,transmembrane or cytoplasmic portions), can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins, which bind to or interact withGL50 (“GL50-binding proteins” or “GL50-bp”) and are involved in GL50activity. Such GL50-binding proteins are also likely to be involved inthe propagation of signals by the GL50 polypeptides or GL50 targets as,for example, downstream elements of a GL50-mediated signaling pathway.Alternatively, such GL50-binding proteins may be GL50 inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a GL50 polypeptideis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aGL50-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the GL50 polypeptide.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a GL50 modulating agent, an antisense GL50nucleic acid molecule, a GL50-specific antibody, or a GL50-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

F. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

GL50 has been mapped to human chromosome 21q22. Accordingly, portions orfragments of GL50 nucleotide sequences (both coding and non-coding),described herein, can be used to correlate these sequences with genesassociated with disease.

The physical position of a sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the GL50 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The GL50 sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the GL50 nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The GL50 nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, 3, or5, can comfortably provide positive individual identification with apanel of primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences are used, a more appropriate numberof primers for positive individual identification would be 500-2,000.

If a panel of reagents from GL50 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial GL50 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the GL50 nucleotidesequences or portions thereof having a length of at least 20 bases,preferably at least 30 bases.

The GL50 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such GL50 probes can be used to identifytissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., GL50 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

G. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining GL50 polypeptideand/or nucleic acid expression as well as GL50 activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant GL50expression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with GL50 polypeptide, nucleic acidexpression or activity. For example, mutations in a GL50 gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby prophylactically treat an individualprior to the onset of a disorder characterized by or associated withGL50 polypeptide, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of GL50 inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of GL50polypeptide or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting GL50 polypeptideor nucleic acid (e.g., mRNA, genomic DNA) that encodes GL50 polypeptidesuch that the presence of GL50 polypeptide or nucleic acid is detectedin the biological sample. A preferred agent for detecting GL50 mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toGL50 mRNA or genomic DNA. The nucleic acid probe can be, for example, ahGL50 nucleic acid, such as the nucleic acid of SEQ ID NO:1, 3, or 5, ora portion thereof, such as an oligonucleotide of at least 15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to GL50 mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

A preferred agent for detecting GL50 polypeptide is an antibody capableof binding to GL50 polypeptide, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect GL50 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of GL50 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of GL50 polypeptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of GL50 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of GL50 polypeptide include introducing into a subject alabeled anti-GL50 antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting GL50 polypeptide, mRNA, orgenomic DNA, such that the presence of GL50 polypeptide, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofGL50 polypeptide, mRNA or genomic DNA in the control sample with thepresence of GL50 polypeptide, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of GL50in a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting GL50 polypeptide or mRNA in abiological sample; means for determining the amount of GL50 in thesample; and means for comparing the amount of GL50 in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectGL50 polypeptide or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant GL50 expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with GL50 polypeptide, nucleicacid expression or activity. Thus, the present invention provides amethod for identifying a disease or disorder associated with aberrantGL50 expression or activity in which a test sample is obtained from asubject and GL50 polypeptide or nucleic acid (e.g., mRNA, genomic DNA)is detected, wherein the presence of GL50 polypeptide or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant GL50 expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) can be administered to a subject to treat a disease ordisorder associated with aberrant GL50 expression or activity. Thus, thepresent invention provides methods for determining whether a subject canbe effectively treated with an agent for a disorder associated withaberrant GL50 expression or activity in which a test sample is obtainedand GL50 polypeptide or nucleic acid expression or activity is detected(e.g., wherein the abundance of GL50 polypeptide or nucleic acidexpression or activity is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberrant GL50expression or activity).

The methods of the invention can also be used to detect geneticalterations in a GL50 gene, thereby determining if a subject with thealtered gene is at risk for a disorder associated with the GL50 gene. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a GL50-protein, or the mis-expression of the GL50gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a GL50 gene; 2) an addition of one or morenucleotides to a GL50 gene; 3) a substitution of one or more nucleotidesof a GL50 gene, 4) a chromosomal rearrangement of a GL50 gene; 5) analteration in the level of a messenger RNA transcript of a GL50 gene, 6)aberrant modification of a GL50 gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a GL50 gene, 8) a non-wild type levelof a GL50 polypeptide, 9) allelic loss of a GL50 gene, and 10)inappropriate post-translational modification of a GL50 polypeptide. Asdescribed herein, there are a large number of assay techniques known inthe art which can be used for detecting alterations in a GL50 gene. Apreferred biological sample is a tissue or serum sample isolated byconventional means from a subject, e.g., a cardiac tissue sample.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the GL50 gene (seeAbravaya et al. (1995) Nucleic Acids Res.23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a GL50 gene under conditions such thathybridization and amplification of the GL50 gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a GL50 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in GL50 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in GL50 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the GL50 gene anddetect mutations by comparing the sequence of the sample GL50 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the GL50 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type GL50 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in GL50s obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a GL50 sequence,e.g., a wild-type GL50 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in GL50 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA: 86:2766, see alsoCotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal.Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample andcontrol GL50 nucleic acids will be denatured and allowed to renature.The secondary structure of single-stranded nucleic acids variesaccording to sequence, the resulting alteration in electrophoreticmobility enables the detection of even a single base change. The DNAfragments may be labeled or detected with labeled probes. Thesensitivity of the assay may be enhanced by using RNA (rather than DNA),in which the secondary structure is more sensitive to a change insequence. In a preferred embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility (Keen et al. (1991)Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner et al. (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a GL50 gene.

Furthermore, any cell type or tissue in which GL50 is expressed may beutilized in the prognostic assays described herein.

VII. Administration of GL50 Modulating Agents

GL50 modulating agents of the invention are administered to subjects ina biologically compatible form suitable for pharmaceuticaladministration in vivo to either enhance or suppress T cell mediatedimmune response. By “biologically compatible form suitable foradministration in vivo” is meant a form of the protein to beadministered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term subject is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Administration of an agent asdescribed herein can be in any pharmacological form including atherapeutically active amount of an agent alone or in combination with apharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeuticcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. For example, a therapeutically active amount of a GL50modulating agent may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability ofpeptide to elicit a desired response in the individual. Dosage regimamay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The GL50 modulating agent (e.g., a peptide, a nucleic acid molecule, oran antibody) may be administered in a convenient manner such as byinjection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active compound may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound. For example,to administer GL50 modulating agent by other than parenteraladministration, it may be necessary to coat the peptide with, orco-administer the peptide with, a material to prevent its inactivation.

A GL50 modulating agent may be administered to an individual in anappropriate carrier, diluent or adjuvant, co-administered with enzymeinhibitors or in an appropriate carrier such as liposomes.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Adjuvant is used in its broadest sense and includes anyimmune stimulating compound such as interferon. Adjuvants contemplatedherein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Sterna et al., (1984) J. Neuroimmunol 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringeability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g., a GL50 polypeptide or anti-GL50 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient (e.g.,peptide) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When the active compound is suitably protected, as described above, theprotein may be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

In one embodiment of the present invention a therapeutically effectiveamount of an antibody to a GL50 polypeptide is administered to asubject. As defined herein, a therapeutically effective amount ofantibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody in the range ofbetween about 0.1 to 20 mg/kg body weight, one time per week for betweenabout 1 to 10 weeks, preferably between 2 to 8 weeks, more preferablybetween about 3 to 7 weeks, and even more preferably for about 4, 5, or6 weeks. It will also be appreciated that the effective dosage ofantibody used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results ofdiagnostic assays as described herein.

Monitoring the influence of agents (e.g., drugs or compounds) on theexpression or activity of a GL50 polypeptide can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase GL50 gene expression, protein levels, or upregulateGL50 activity, can be monitored in clinical trials of subjectsexhibiting decreased GL50 gene expression, protein levels, ordownregulated GL50 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease GL50 gene expression,protein levels, or downregulate GL50 activity, can be monitored inclinical trials of subjects exhibiting increased GL50 gene expression,protein levels, or upregulated GL50 activity. In such clinical trials,the expression or activity of a GL50 gene, and preferably, other genesthat have been implicated in a disorder can be used as a “read out” ormarkers of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including GL50, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates GL50 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on a GL50 associated disorder, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of GL50 and other genes implicated in the GL50associated disorder, respectively. The levels of gene expression (i.e.,a gene expression pattern) can be quantified by Northern blot analysisor RT-PCR, as described herein, or alternatively by measuring the amountof protein produced, by one of the methods as described herein, or bymeasuring the levels of activity of GL50 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a GL50 polypeptide,mRNA, or genomic DNA in the pre-administration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the GL50 polypeptide, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the GL50 polypeptide, mRNA, or genomic DNAin the pre-administration sample with the GL50 polypeptide, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of GL50 to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of GL50 to lower levels than detected,i.e. to decrease the effectiveness of the agent. According to such anembodiment, GL50 expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES

The following materials and methods were used in the examples.

Mouse strain and RNA isolation: Mice (C57B1/6) injected with 10E5 MB49bladder carcinoma cells were treated with 1 μg/mouse recombinant IL12 ondays 7-11 and 14-18. RNA was isolated from lymph nodes on days 9 (75%),12 (20%) and 19 (5%) were subsequently pooled. RNA was extracted usingRNAStat 60 (teltest B) followed by poly A+ RNA enrichment using polyAttract magnetic isolation system (Promega). cDNAs were synthesized withsuperscript RT (Gibco BRL). Additional cDNA sources include a mousefetal thymus library (C3H/Hej) and mouse peripheral blood lymphocytesderived from cardiac puncture of C57B1/6).

Signal trap: Signal trap protocols were followed as described by Jacobset al. (1997. Gene. 198: 289). Briefly, size fractionated cDNAs wereunidirectionally cloned into the invertase expression plasmidpSUC2T7M130RI. An expression library of plasmid clones was generated inE. coli and subsequently introduced into the invertase deficientsuc2-strain of yeast. Signal trapped clones represented in the yeastlibrary were selected by 2 day culture in YPR agar plates. Three hundredand thirty three clones were picked at random, miniprepped andsequenced.

Sequence analysis: TBlastX, FastX, pFam, Pileup, GrowTree and Sigcleaveof GCG Wisconsin package, and Geneworks 2.5.1 was used for DNA sequencemanipulation, database searching and sequence analysis. In FIG. 12,identity scores for pileup analysis was determined according to thefollowing values: 1× pair=1; 2× pair=2; 3× pair=3; 3-of-a-kind=4;3-of-a-kind plus 1× pair=5; 2× 3-of-a-kind=6; 4-of-a-kind=7; 4-of-a-kindplus 1× pair=8; 5-of-a-kind=9. The Lasergene DNAstar Genequest modulewas used for delineating intron-exon boundaries of hGL50 against GenbankAccession #HS21C098. Further analysis was performed with the SeqWebWisconsin GCG package using TFASTA, TBLASTN, ProfileScan.Distance-proportional phylograms were generated by GrowTree based ongenetic distance using Kimura correction algorithms. Graphical outputwas subsequently reformatted to reflect family clusters.

3′ rapid amplification of cDNA ends: 3′ RACE was performed using primers(GL50) VL118 (CCCGCAGTCTGCGCTCGCACC; SEQ ID NO:7), VL116(GTCGACCCACCATGCAGCTAAAGTGTCCCTG; SEQ ID NO: 8), (AB014553) VL141(CGTGTACTGGATCAATAAGACGG; SEQ ID NO:9), VL142 (ACAACAGCCTGCTGGACCAGGC;SEQ ID NO:10), (Poly A-oligo) VL054 (CCAGTGAGCAGAGTGACG; SEQ ID NO:11),VL055 (GAGGACTCGAGCTCAAGC; SEQ ID NO:12). Mouse peripheral bloodlymphocytes (PBLs) were enriched for lymphocytes by densitycentrifugation using lympholyte M according to the manufacturer'sprotocol. Human PBLs were isolated by Ficoll-paque densitycentrifugation of human leukopac samples. Total RNA was extracted fromlymphocytes as described below. Reverse transcription was accomplishedusing primer VL053 (CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTT; SEQID NO:18), 5 μg of total RNA and SuperScript RT (Gibco-BRL) according tothe manufacturer's protocols in 20 μl reactions. 0.5-1.0 μl ofRT-synthesized cDNAs were used per RACE procedure. 3′ RACE was performedaccording to the method of Frohman, M. A. (1993) Methods Emzymol.218:340-356.

RNA isolation and analysis: Total RNA was derived from CCE ES cells,Swiss Webster embryos/yolk sacs and C57B1/6 peripheral blood lymphocytesand was extracted using RNAstat 60 (Tel-Test B, Friendswood Tex.)accompanied with Phase-lock gel barrier (Eppendorf). RNA wasfractionated using Northern Max system (Ambion) and blotted ontoZetaProbe GT (BioRad) according to the manufacturer's protocols.Multiple tissue RNA panels were purchased (Clontech) and used accordingto the manufacturer's instructions. Blots were hybridized toradiolabeled DNA fragments encompassing either nucleotides 984-1340 ofthe mGL50-2 clone (357 bp; SEQ ID NO:3), corresponding to the 3′untranslated region, while fragments corresponding to the codingsequence of mGL50 were used to detect both mGL50-1 and mGL50-2transcripts. Hybridizations were performed at 65° C. with Express Hyb(Clontech) overnight and subsequently washed with 0.1×SSC and 1% SDS athybridization temperatures until a suitable signal to noise ratio wasreached. Blots were exposed to phosphoimage plates and autoradiographicfilm for imaging.

Gene expression analysis: For RT-PCR analysis, first strand cDNAsynthesis was performed as described above for RACE procedures, followedby duplicate 25 μl amplification reactions (using Advantage Taq,Clontech) with the primers RLEE 001 and RLEE005 for mGL50-1 and primersRLEE 001 and RLEE003 for mGL50-2. Primers GAPDH-F and GAPDH-R were usedas positive amplification controls. The oligonucleotides GAPDH-F(TGAAGGTCGGTGTGAACGGATTTGGC; SEQ ID NO:19); GAPDH-R(CATGTAGGCCATGAGGTCCACCAC (SEQ ID NO:20); RLEE001(CATCACTAGCATTAGCCAGGC; SEQ ID NO:13); RLEE003 (TGATGTTGTGAAGCTGAGTGC;SEQ ID NO:14); RLEE005 (TCATGAGCATCGAGCATCG; SEQ ID NO:15); VL142(ACAACAGCCTGCTGGACCAGGC; SEQ ID NO:10); VL162B(TCACGAGAGCAGAAGGAGCAGGTTCC; SEQ ID NO:16); and VL163B(GGGCCCCCCAGAACCTGCTGCTTCC; SEQ ID NO:17) were designed for the PCRamplification of the extracellular domain regions of mGL50-1, GL50-RACE,AB014553 cDNA and AB014553-RACE clones. Mouse and human cDNA panelsderived from poly A+ RNA encompassing lymphoid and nonlymphoid tissues(Clontech) was used as a source for PCR analysis. Cycling conditionswere 5 min 95° C. denaturing step followed by 35 cycles of 1 min at 95°C., 1 min at 60° C., and 1 min at 72° C. The reaction was terminatedfollowing a 10 min 72° C. extension. Cycling conditions for mGL50 andmGL50-2 PCR were 95° C. for 1 min, 60° C. for 1 min, and 72° C. for 2min. for 33 cycles, while GAPDH PCR was performed using 30 cycles.

For Northern blot analysis, commercially prepared RNA blots (Clontech)were hybridized to radiolabeled DNA fragments encompassing nucleotides1065-1588 of mGL50-1 (494 bp; SEQ ID NO:1), or nucleotides 984-1340 ofthe mGL50-2 clone (357 bp; SEQ ID NO:3).

Flow cytometry: COS cells were transfected with mGL50-1 or DAP-12 cDNAin pcDNA3.1-CTGFP expression vectors. Transfection was accomplishedusing lipofectamine transfection reagent (Life Technologies) accordingto manufacturer's protocols. Cells were harvested 3 days aftertransfection. 10% Rabbit serum was used to block non-specific binding tocells. Cells were stained at room temperature for 20 minutes with 200 ngof fusion proteins in 100 μl of PBS 2% FCS. Cells were washed andsecondary staining performed with PE-linked goat anti-mouse IgG. Cellswere stained with propidium iodide immediately prior to flow cytometry.Positive COS transfection control was performed with hCTLA4 cDNAfollowed by identification of positively staining cells with PE-linkedanti CTLA4.

Cell suspensions for cytometric analysis were isolated from Balb/csplenocytes (˜3 months old) and washed once with DMEM, 10% (vol/vol)heat-inactivated fetal calf serum (JR BioScience), 2 mM L-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin (Irvine Scientific, Santa Ana,Calif.), 20 μM 2-betamercaptoethanol (Sigma Co., St. Louis, Mo.), MEMsodium pyruvate, and MEM Non-essential amino acids (Life Technologies,Rockville, Md.). Red blood cells were lysed with ACT lysing buffer andwashed once. Splenocytes (˜1×10 ⁷ cells/ml/well) from Balb/c mice werecultured with 25 μg/ml LPS (Sigma) or 10 ng/ml PMA, 1 μg/ml ionomycin.Cells were stained with FITC-labeled antibodies (BD-Pharmingen) andmICOS-mIgG2am reagent, followed by flow cytometric analysis using theFACalibur and CellQuest software package (BD). Cell separation wasperformed using anti-FITC microbead magnetic selection (Miltenyi Biotec)followed by flow cytometric determination of T-cell enrichment.

Ig fusion proteins: Fusion proteins of IgG2a with mICOS, hICOS, mGL50-1,and hGL50 were constructed for use in the following examples. Thenotation IgG2am indicates that the IgG2a domain was mutated to reduceeffector function (as in Steurer, W. et al. (1995) J. Immunol.155:1165-74). The nucleotide and amino acid sequences of hICOS-mIgG2amare presented in FIG. 26 and set forth as SEQ ID NOs:23 and 24,respectively. The nucleotide and amino acid sequences of mICOS-mIgG2amare presented in FIG. 27 and set forth as SEQ ID NOs:25 and 26,respectively. The nucleotide and amino acid sequences of hGL50-mIgG2amare presented in FIG. 28 and set forth as SEQ ID NOs:27 and 28,respectively. The nucleotide and amino acid sequences of mGL150-mIgG2amare presented in FIG. 29 and set forth as SEQ ID NOs:29 and 30,respectively.

Example 1 Isolation of mGL50-1 Molecules

cDNAs encoding secreted proteins derived from RNA of IL-12 treated mouselymph nodes were placed under genetic selection for signal sequences byusing the Saccharomyces cerevisiae signal sequence trap method (Jacobset a). Of a total of 333 cDNA:invertase clones isolated and sequenced, 1partial cDNA clone with limited sequence identity with B7-1 wasidentified and termed mGL50-1 (FIG. 1, SEQ ID NO:1). RecA mediated fulllength cDNA isolation from a mouse fetal thymus cDNA library resulted inthe generation of 4 additional cDNA clones that contained 3′untranslated regions as well as overlapping the partial signal trappedsequence clone.

The consensus 2718 nucleotide mGL50-1 sequence encoded a 322 amino acidprotein with a predicted mass of 36 kDa. Hydropathy plot of the openreading frame predicted a structure corresponding to a leader sequence(from about amino acids 1-46 of SEQ ID NO:2; encoded by aboutnucleotides 67 to 195 of SEQ ID NO:1), an extracellular domain (fromabout amino acids 47-279 of SEQ ID NO:2; encoded by about nucleotides196 to 904 of SEQ ID NO:1), a hydrophobic transmembrane region (fromabout amino acids 280-298 of SEQ ID NO:2; encoded by about nucleotides905 to 961 of SEQ ID NO:1) and a potential intracellular cytoplasmicdomain (from about amino acids 299-322 of SEQ ID NO:2; encoded by aboutnucleotides 962 to 1032 of SEQ ID NO:1). Signal peptide cleavage waspredicted at position 46 in the amino acid sequence. Analysis of mGL50-1by Pfam protein motif prediction program suggested structural similarityto Ig-domain in the cytoplasmic domain of the protein. In keeping withan Ig-like structure, 4 cysteines were found in the extracellulardomain, allowing for the possibility of intramolecular bonding anddistinct structural conformation corresponding to an IgV-like domain andan IgC-like domain, based on domain delineation. FastX sequencecomparison in which translated proteins are searched through GenBankdatabase yielded a number of identified cDNA clones with sequencesimilarities including AB014553, B7-1, B7-2, and Y08823. Correspondingdomains in polypeptides in the B7 family are shown in FIG. 12.

Example 2 Isolation of an Alternatively Spliced Form of GL50

To determine the extent of transcript heterogeneity, 3′ RACE wasperformed to isolate splice variants of murine GL50-1. Using specific,nested 5′ oligonucleotide primers corresponding to sequences upstreamand including the initiation start site of mGL50-1, amplified PCRproducts were generated from cDNAs derived from mouse PBLs. Uponhybridization to radiolabeled oligonucleotides internal to mGL50-1coding region, clear hybridization signals were detected. Subsequentcloning of positively hybridizing PCR products followed by sequenceanalysis revealed RACE sequences of which none were identical to theconsensus mGL50-1 sequence derived from the mouse fetal thymus library.Two sets of PCR products, represented by multiple clones with extensivepolyadenylation of differing lengths, were found to encode analternatively spliced form of GL50. One representative product, a 1759bp product, termed mGL50-2, encoded a polypeptide 347 amino acidresidues in length with a predicted molecular mass of 39 kDa (FIG. 2,FIG. 15).

An alignment of mGL50-1 and mGL50-2 is presented in FIG. 3. Alignment ofthe mGL50-1 and mGL50-2 sequences demonstrated complete identity fromnucleotide 67 (initiation methionine/mGL50-2 RACE priming site) tonucleotide 1027 of the cDNA, with the exception of two nucleotides foundin multiple mGL50-2 products (nucleotides 531 and 710, leading to anarginine to histidine residue at 237 of the predicted amino acidsequence (FIG. 3)). These two nucleotide discrepancies are most probablydue to strain differences between the mice used for the RNA startingmaterial, since multiple separate PCR products encoded identicalmismatches. Sequences downstream of position 1027 of mGL50-1 andposition 961 of mGL50-2 were divergent between the two molecules (FIG.3). Both mGL50-1 and mGL50-2 sequences contained a consensus AATAAApolyadenylation signal upstream from the poly-A tail (13 bp for mGL50-2,16 bp for mGL50-1). As a result of the alternative 3′ sequences encodingthe carboxy terminus, mGL50-2 lacked the final 2 amino acids of mGL50-1but incorporated an additional 27 novel amino acids in the cytoplasmicdomain. The predicted amino acid sequence of mGL50-2 indicated thepresence of three unique tyrosine residues, Y325, Y328, and Y333, in thecarboxy terminus, in addition to the tyrosine residues Y299 and Y307shared by both the mGL50-1 and mGL50-2 molecules. GenBank databasesearch revealed no cDNA sequences with similarity to the divergentcoding 3′ domain of the mGL50-2 product, with the exception of a complexrepetitive sequence (bases 1349-1554) also found in numerous genomicsequences (e.g. Accession numbers AC005818, AC006508, and AF115517), aswell as in known mRNAs (mouse desmin: Z18892; and mouse servivin:AF115517). No such untranslated repetitive sequences were found inmGL50-1.

Example 3 Identification of a Human Ortholog of GL50

After the murine GL50 clones were identified, database search andsubsequent comparisons suggested that mouse mGL50-1 and mGL50-2 clonesmay have homology with a cDNA isolated from human brain, KIAA clone 0653(accession # AB014553; Ishikawa et al. (1998) DNA Res. 5:169). AB014553has been described as a 4.3 kb cDNA localized on chromosome 21, encodinga putative 558 amino acid protein with a molecular mass of 60 kDaBecause both the length of the AB014553 cDNA and the encoded proteinwere nearly 2 fold greater than mGL50-1, it was not likely that AB014553was a human ortholog of the mouse GL50 sequences. However, analysis ofthe first 303 residues of the deduced AB014553 protein sequenceindicated similarity with mGL50-1, excluding the signal peptide regionof the cDNA.

Because AB014553 was derived by size fractionation of large cDNAs,AB014553 was believed to represent a variant transcript that alsoexisted as a smaller gene product. To address whether such a smallerproduct existed, 3′ RACE analysis of human PBLs with oligonucleotidesprimers (VL142 (ACAACAGCCTGCTGGACCAGGC; SEQ ID NO:10) and VL141(CGTGTACTGGATCAATAAGACGG; SEQ ID NO:9)) corresponding to extracellulardomains of AB014553 with sequence homology with GL50 were performed.Four RACE products were isolated which encoded an open reading frameidentical to AB014553 from amino acid residue 24 (starting point of RACEprimer) to residue 123 (FIG. 6). From residue 123 onward, the AB014553RACE product diverged from the cDNA sequence resulting in an alternative88 nucleotides with a 3′ coding region encoding 9 amino acids,termination codon, and a short untranslated domain. This alternative 3′region resulted in a premature termination codon in the AB014553 RACEclone as compared to AB014553 cDNA (FIG. 7). The predicted total lengthof the deduced polypeptide encoded by this alternatively transcribedproduct, after merging with shared 5′ sequences of AB014553 cDNA was 309amino acids, consistent with a human protein orthologous to mouse GL50protein sequences, referred to as hGL50 (FIG. 8).

Example 4 Alignment with Chicken B7-1

Upon alignment with a previously characterized chicken B7-1 (AccessionNo. Y08823), a pattern of conserved cytoplasmic domain sequences emergedbetween these molecules. Within the intracellular region, hGL50 proteinsequences exhibited 34% identity (9/26 residues aligned) with mGL50-1,while chicken Y08823 exhibited 57% identity (8/14 residues aligned) witheither human or mouse GL50 or GL50-2 resulting in a consensus motif of(R)(R)(R)[XX](W)(H)(X/-)SY(T)(G)(P) (SEQ ID NO:21), wherein amino acidsin brackets are variable between the three genes, amino acids inparentheses are shared between two of the three genes, and amino acidswithout brackets or parentheses are shared by all three genes. A FastAdatabase search for proteins with homology to this motif yielded twomouse entries, Veli-2 (Accession No. AF087694) and MALS-2, a C. elegansLIN-7 homolog (Accession No. AF173082), encoding identical sequenceswith the motif RRRQQHHSYT (SEQ ID NO:22). This unique domain islocalized at the carboxy terminus of Veli-2 but is not present in theisoforms Veli-1 or Veli-3, and extends beyond the area of homology withC. elegans LIN-7.

Example 5 Expression of GL50 Molecules

mGL50-1 and mGL50-2 specific RT-PCR reactions on commercial cDNA panelsresulted in abundant PCR products generated in heart, spleen, lung,liver, skeletal muscle, kidney, testis, 7-15 day embryo and PBL.Negligible product was detected with brain samples for eithertranscripts while low levels of product was detected in testis samplesfor mGL50-2 (FIG. 4). By Northern blot analysis of commercial RNA blotsusing probes specific to either the shared extracellular domain ofmGL50-1 and mGL50-2 or to the 3′ untranslated regions of either mGL50-2or mGL50-1, differential hybridization was found between the twomolecules. Whereas both the extracellular domain probe and the mGL50-1specific probe hybridized to an ˜2.7 kb message clearly detectable inheart, brain, spleen, lung, liver, skeletal muscle, kidney and testissamples (identical to the pattern previously seen in blots specific formGL50-1 (Ling et al. (2000) J. Immunol. 164:1653-7), the mGL50-2specific probe hybridized to a 1.7 kb transcript detected only in heart,spleen and kidney samples, suggesting that mGL50-2 transcripts wereconcurrently transcribed as a limited subset of tissues with the highestexpression mGL50-1 (FIG. 5). In poly A+ RNA blots, hybridization usingthe mGL50-2 3′ UTR specific probe was clearly detected in samplesrepresenting undifferentiated ES cells, day 10 embryoid bodies, day 12.5embryonic yolk sac, and day 15 fetal liver. In contrast, hybridizationusing the mGL50-1 cDNA coding sequence probe clearly revealed transcriptin all samples examined

To assess the tissue distribution of AB014553 cDNA and AB014553 RACEclones, RT-PCR/southern blot analyses were performed under similarconditions as for the GL50 sequences described above. Usingoligonucleotides primers specific for the amplification of publishedAB014553 cDNA (VL142 (ACAACAGCCTGCTGGACCAGGC; SEQ ID NO:10) and VL163B(GGGCCCCCCAGAACCTGCTGCTTCC; SEQ ID NO:17)), PCR resulted in the completeabsence of any detectable AB014553 cDNA signal for all samples tested(FIG. 10). Possible explanations for the lack of RT-PCR productsrepresenting published AB014553 cDNA sequences may be the use ofnon-optimized oligonucleotides, extremely low abundance of the targettranscript, or actual absence of this form of the product. RT-PCRconditions specific for AB014553 RACE using oligonucleotide primersVL142 and VL162B resulted in the detection of a 350 bp amplificationproduct in kidney, lung, ovary, fetal liver, and leukocyte, with thehighest level of amplified product detected in fetal liver.Surprisingly, virtually no signal was detected in spleen, lung, thymus,or lymph nodes. These results are consistent with the published reportof AB014553 transcript distribution (Ishikawa et al. (1998) DNA Res.5:169) in a smaller survey of a tissue cDNA panel, but does notcomplement the tissue distribution patterns observed for the GL50molecules.

Unlike the mGL50-1 and mGL50-2 clones in which lengthy and divergent 3′untranslated regions were present, AB014553 RACE products contained only88 bp of sequence that diverged from that of AB014553 cDNA. Because ofthis, it was not possible to design nucleotide probes of sufficientspecific activity for the detection of the RACE product. Using codingregion probe for hGL50 northern hybridizations were performed oncommercial human multiple tissue RNA blots to assess transcriptdistribution (FIG. 11). Results indicated the presence of a number oftranscripts found in all tissues with approximate molecular size of 2.4kg, 3.0 kb, 7.0 kb, with highest levels of signal present in brain,heart, kidney, and liver samples. Low hybridization signals weredetected in colon and thymus. An additional transcript of 8.5 kb wasdetected in a subset of the panel, including thymus, spleen, kidney,liver, lung and PBL while a 3.8 kb transcript was detected in lung andPBL sample. A unique 1.1 kb transcript was detected only in PBL samplesand corresponded to the predicted size of hGL50 if 5′ and 3′untranslated sequences were included. Determination of other minortranscripts was difficult due to the limits of the sensitivity range ofthe blot. None of the obvious transcripts correlate with the 4.3 kbpublished AB014553 cDNA, suggesting that this sequence may not exist innature or may be expressed at levels lower than detectable limits.Comparison between hGL50 blots and hGL50 RT-PCR surveys share the commonfeature of having the greatest signal in kidney tissues and less signalin lymphoid related tissues such as thymus, spleen and PBL.

Example 6 Relationship of the GL50 Polypeptides to Other Polypeptides

To determine the extent of relatedness between mGL50-1, hGL50, and humanand mouse B7-1 and B7-2, protein sequence alignments were performed.From Pileup analysis (FIG. 12), 18 amino acid locations alignedidentically between all six molecules within the extracellular domain.Of the 32 positions that define the predicted IgV-like and IgC-likefolds of the B7 molecule, 13 are identically conserved between all sixmolecules, most notably the 4 cysteines that allow intramolecularfolding of domains. Other areas of significant sequence conservationwere also seen in the extracellular domain, but interestingly theidentities of hGL50/mGL50 sequences in certain locations aligned moreclosely with either B7-1 or B7-2 (identity score of 8). For example,valine residue corresponding to position 77 of mGL50-1 is shared byhGL50, and murine and human B7-2 sequences, but not B7-1. Likewise, thetyrosine at position 78 of mGL50-1 is conserved at correspondinglocations in hGL50 and murine and human B7-1, but not B7-2. Of the 16positions with identity scores of 8, 5 positions are shared bymGL50-1/hGL50 and B7-1, 4 positions are shared between mGL50-1, hGL50and B7-2, and 6 positions are shared between B7-1 and B7-2.

Based on the peptide structure, these results suggest that themGL50/hGL50sequences occupies a phylogenetic space parallel to the B7family of proteins. Molecular phylogeny analysis (GrowTree) measuringgenetic distance in terms of substitutions per 100 amino acids resultedin a dendrogram (FIG. 13) with independent clustering of mGL50/hGL50(85), m/hB7-2(68) and m/hB7-1 (88). As an outgroup, mmu67065_(—)1 (mousebutyrophilin) was used. The chicken clone Y08823 also was found to bemore aligned with the GL50/AB014553 sequences (˜140) than the B7sequences (215-320), indicating that these sequences comprised adistinct subfamily of proteins. Distances between the GL50/AB014553,B7-2 and B7-1 branches were high (216-284), suggesting that largenumbers of substitutions have occurred between these molecules since theinception of the human and rodent lineage.

Mouse and human CTLA4 (see e.g., Dariavach, P. et al. (1988) Eur. J.Immunol. 18:1901; GenBank Accession Number L15006; U.S. Pat. No.5,434,131) and ICOS (Hutloff et al. (1999) Nature 397:263; WO 98/38216)were also analyzed for phylogenetic relationships using the sameparameters. Genetic distances revealed a pattern that was distinct tothat seen for the B7-like proteins. As indicated in previous reports,the genetic distance between the mouse and human ICOS and CD28(176-2570) was closer than that of CTLA4 (261-405). By comparison, thegenetic distance between CD28 and CTLA4 was much smaller (143-1670),indicating that the structural relationships between the members of thereceptor family were not parallel to that of the ligand family.

Example 7 Demonstration of Binding of GL50 to ICOS

To determine whether GL50 was a ligand for murine CTLA4, CD28 or ICOS,transfection binding studies were performed with mGL50-1 expressionvectors (FIG. 14). mGL50-1 or human DAP-12 negative control cDNA weretransfected into COS cells followed by staining with either ICOS-Ig,CD28-Ig or CTLA-4-Ig fusion proteins or normal murine Ig. COS cells werestained two days after transfection with 5 μg/ml of fusion protein,followed by goat anti-mouse PE labeled antibody. By flow cytometry,binding of GL50 transfected COS cells was detected only by the ICOS-Igreagent (15%), while negligible binding was detected for CD28-Ig,CTLA4-Ig or the normal mouse Ig used as a negative staining reagent. Nobinding of any fusion protein was detected for the DAP-12 cDNAtransfectants. These results suggest that GL50 is a ligand for ICOS-Ig.

Although not found under the specific binding conditions herein, it maybe that GL50 is also capable of signaling through either CD28 or CTLA-4given the published data showing the weaker binding activity of the B7molecules to CD28 than CTLA-4 (Greenfield, E. A. et al. (1998) Crit.Rev. Immunol. 18:389) in cell based assays.

Example 8 mGL50-2 Transcripts Encode Functional Cell Surface Proteins

To demonstrate that mGL50-2 transcripts encode functional cell surfaceproteins, vectors expressing the mGL50 coding regions under thetranscriptional control of EF-1alpha promoter were used to transfect COScells. By flow cytometry, both mICOS-mIgG2am and hICOS-mIgG2am werefound to bind mGL50-1 and mGL50-2 transfected cells (9-14%) whilenegligible binding was observed with mCTLA4-mIgG2am (<1%), indicatingthat the domains encoded by the additional residues in the alternatecarboxy-tail found in mGL50-2 do not affect surface mobilization of thisprotein (FIG. 17). It is also notable that hICOS-mIgG2am binds bothmolecules, suggesting that the ICOS receptors, like CTLA4 and CD28receptors, retain ligand binding capacity when assayed against targetsacross primate/rodent species boundaries. Other mouse cells lines wereexamined for the presence of surface ICOS-ligand. WEHI231cells have beenpreviously shown to have surface expression of both B7-1 and B7-2,whereas ES cells have been shown to display only B7-1. mCTLA4-mIgG2amstaining of WEHI 231 cells was clearly detectable using 8 ng/ml ofreagent, while mICOS-mIgG2am staining was minimally detectable at levelsstarting at 1 μg/ml. These results suggest that the binding affinity ofmCTLA4-mIgG2am reagent to the B7 molecules is at least 100 fold greaterthan mICOS-mIgG2am reagent binding to GL50 on WEHI cells, similar to thelow binding affinity measured between CD28-Ig and B7 proteins. In thepresence of blocking antibodies, mCTLA4-mIgG2am binding to WEHI 231 wastotally abrogated, while no effect on mICOS-mIgG2am binding to cells wasobserved, confirming that neither WEHI 231 B7-1 nor B7-2 potentiatesspecific binding with mICOS-mIgG2am (FIG. 18). To corroborate evidencefrom RNA blot analysis demonstrating the presence of GL50 in cellsrepresentative of the very early embryonic environment (see above),undifferentiated CCE ES cells were analyzed by direct staining withantibodies to B7-1 and indirect staining with mICOS-mIgG2am fusionprotein. Undifferentiated ES cells stained with anti-B7-1 (FIG. 19)revealed a one-log fluorescence shift over background, consistent withprevious observations (Ling, V. et al. (1998) Exp. Cell. Res.241:55-65), and a half-log fluorescence shift over background withmICOS-mIgG2am staining, demonstrating the simultaneous surface displayof both B7 and GL50 type molecules in a system that reflects theundifferentiated inner cell mass of early preimplantation embryos.

Example 9 Expression of GL50 on Splenocyte Subpopulations

Phenotypic analysis of the major splenic cell types exhibiting GL50surface proteins revealed mICOS-mIg binding to be most readilydetectable on phenotypic CD19+ B cells, although it was apparent thatother splenic cell types exhibited ICOS-Ig staining (see FIG. 30). Tofurther identify other freshly isolated cells that display GL50, wildtype Balb/C splenocytes were compared to RAG1 −/− splenocytes lackingmature B and T cells. The results are presented in FIG. 20 and Table 3.TABLE 3 Balb/C RAG1-/- Antibody % of Total % ICOS-Ig % of Total %ICOS-Ig stain n = Splenocytes positive n = Splenocytes positive anti-CD310,000 30% 10% 50,000 <1% — anti-CD4 10,000 25%  8% 10,000 11% 45%anti-CD8a 10,000  9% 10% 50,000 <1% — anti-CD19 10,000 65% 97% 50,000<1% — anti-CD24 10,000 64% 94% 10,000 67% 28% anti- 10,000 61% 97%50,000  6%  5% CD45R/B220 anti-CD11B 50,000  8% 26% 10,000 37% 31%anti-CD11C 50,000  2% 43% 10,000 20% 55% anti-pan NK 50,000  3% 20%10,000  9%  3% anti class II 10,000 65% 95% 10,000 27%  3% anti CD4010,000 61% 97% 10,000 <1% — anti CD69 10,000  2% 25% 50,000  3%  5%

As expected, Balb/C splenocytes revealed high levels of mICOS-mIgG2ambinding (FIGS. 20A and B) to phenotypic B cells (CD19, B220, CD40 >94%),while lower levels were found on phenotypic T cells and T cell subsets(CD3+, CD4+, and CD8+; <10%), macrophages (CD11b, 26%), dendritic cells(CD11c, 43%) and NK-cells (pan-NK, 20%). mICOS-mIgG2am binding was alsodetected on the more general lymphoid markers CD24 and class II (94%)cells. Northern blot analysis (using an mGL50-1 specific probe)demonstrated that GL50 transcripts are expressed in the splenocytes ofRAG1 −/− mice. This suggested that in the absence of mature T or Bcells, GL50 was still expressed on other splenocyte subpopulations.Consistent with these observations, analysis of RAG1 −/− splenocytes(FIG. 20B) demonstrated that they are CD3−, CD8−, CD19−, and CD40−, andthat the remaining CD11b+ (35%) and CD11c+ (55%) cells are readilycounterstained with mICOS-mIgG2am. Low levels (<5%) of ICOS-Ig stainingwere also apparent in B220+, panNK+, and CD69+ cells. It is notcurrently understood why there is a disparity in mICOS-mIg staininglevels between these three markers on RAG1 −/− splenocytes, whencompared with the higher levels detected in Balb/c splenocytes.mICOS-mIgG staining of CD4+ (45%) and CD24+ (28%) cells was alsoapparent in RAG1 −/− splenocytes despite the absence of staining forother T cell markers. CD4+ staining has previously been reported ondendritic cells (Aicher, A. et al. (2000) J. Immunol. 164:4689-96), andthis was supported by the presence of a CD4+, CD11c+ double positivecell population in these mice (FIG. 20C). The presence of GL50transcripts in conjunction with mICOS-mIgG binding of phenotypicmacrophage and dendritic cell subsets in RAG1 −/− splenocytes verifiesthe presence of ICOS-ligand on professional antigen presenting cellsthat may potentiate signaling through ICOS in vivo.

Example 10 Expression of GL50 Splice Variant mRNAs in SplenocyteSubpopulations and Embryonic Cells

Because ICOS-ligand appeared to exist as at least two splice variants,experiments were performed to semi-quantitatively assess the presenceGL50-1 and GL50-2 transcripts in splenocyte cell populations. Balb/Csplenocytes cultured in the presence of LPS or ConA were found toupregulate ICOS-ligand in all splenocytes examined (FIG. 20). Todetermine if preferential stimulation of these cells caused differentialupregulation of GL50-1 or GL50-2 transcripts, GL50-1 and GL50-2transcripts were detected by RT-PCR using transcript specificoligonucleotide primers and hybridization probe sets. The results arepresented in Table 4. TABLE 4 RT-PCR Analysis of mGL50 Isoforms Balb/Cw.t. LPS ConA mGL50 mGL50-B GAPDH mGL50 mGL50-B GAPDH mGL50 mGL50-BGAPDH Spleen + + (+) + + (+) + +/− (+) CD4 + + (+) + +/− (+) + +/− (+)CD8 − − (+) +/− − (+) + +/− (+) CD19 ++ ++ (+) ++ ++ (+) + − (+) RAG-1-/- Spleen + +/− (+) RAG-1 -/- CD11c + + (+) RAG-1 -/- CD11b + +/− (+)F5M + − (+) F5M LPS + +/− (+) WEHI 231 + +/− (+) D0 ES cells ++ ++ (+)D11.5 Embryo + + (+) D12.5 Embryo ++ + (+) D11.5 Yolk ++ + (+) Sac WaterControl − − −Amplifications were performed in duplicate followed by autoradiographicdetection of blotted GL50 samples.− represents the absence of signal in duplicate samples.+/− represents the presence of signal in one of the duplicate samples.+ represents the presence of signal within both members of duplicatesamples.++ represents autoradiographic saturation of signal within duplicatesamples.(+) represents visual detection of amplified GAPDH products by ethidiumbromide staining

Balb/C CD4+, CD8+ and CD19+ cell subsets and RAG1 −/− CD11b+ and CD11c+cell subsets were enriched to >90% purity by bead separation. DuplicateRT-PCR analyses of quantity-normalized RNA samples revealed GL50-1 andGL50-2 transcripts to be present in non-treated CD4+ T-cells and CD19+ Bcells, consistent with results from flow cytometric analysis. However,neither GL50-1 nor GL50-2 transcripts were amplified in CD8+ T cells,despite surface protein detection by FACS and enrichment of ICOS-ligandpositive cells. It is possible that CD8 GL50 expression is below thethreshold of detectability by RT-PCR, or that CD8+ ICOS-ligand is yetanother variant of GL50 not targeted for detection by this assay. Also,one cannot rule out the possibility that the form of ICOS ligandappearing on CD8+ cells may not be GL50-1 or GL50-2, as describedherein, or that the CD8+ ICOS ligand may originate elsewhere as asoluble protein and become transferred to this cell type. LPS activationled to a profile similar to that seen for control cells, with theexception that low levels of GL50-1 were detected in CD8+ samples,suggesting that LPS stimulation of B cells may indirectly upregulateexpression of this form of ICOS-ligand on T cells. ConA stimulation ofsplenocytes resulted in the amplification of GL50-1 transcripts acrossall samples with a decrease of product in CD19+ cells. GL50-2transcripts were induced in CD8+ samples and were not detected in CD19+samples. The decrease of amplified product of both GL50-1 and GL50-2 inCD19+ cells suggests a regulation of B cell transcription upon exposureto ConA. In RAG1 −/− splenocytes, GL50-1 and GL50-2 were detected inCD11b+ and CD11c+ positive cells, while cultured dendritic F5M andWEHI231 cells exhibited GL50-1 transcripts. Low levels of GL50-2 weredetected in WEHI 231 and LPS activated F5M cells, while no amplifiedproduct was detected in uninduced F5M cells. In samples representingembryonic tissues, GL50-1 and GL50-2 were detected in all samples, withabundant levels of both splice variants present on D0 ES cells. Highlevels of GL50-1 were also detected in day 12.5 embryo and 11.5 yolk sacsamples. These results correlate with the degree of transcripthybridization shown by RNA blot analysis (see above).

Example 11 The Chicken GL50-Like Molecule Y08823 Does Not Bind ICOS

Very recently, the crystal structure of B7-1 was resolved at the threeangstrom level, revealing a structure comprised of parallel, 2-foldrotationally symmetric homodimers with charged residues in theamino-terminal domain of B7-1 responsible for direct interactions withCD28/CTLA4. Human and mouse GL50, B7-1, and B7-2 protein sequencesexhibit 19-27% sequence identity (Table 5) suggesting that they may alsoshare structural similarities. TABLE 5 Alignment scores between GL50,B7-1, and B7-2 related proteins Percent Sequence Identity hGL50 Y08823mGL50 mGL50-B hB7-2 mB7-2 hB7-1 mB7-1 Genetic hGL50 — 36 44 44 19 24 2522 Distance Y08823 138 — 37 37 28 23 26 30 mGL50 85 131 — 99 24 25 24 27mGL50-B 85 131 0.4 — 26 23 26 26 hB7-2 270 230 221 221 — 51 26 30 mB7-2251 310 200 200 68 — 24 28 hB7-1 243 224 247 247 222 243 — 45 mB7-1 261223 282 282 190 182 88 — mmu67065 188 219 214 214 207 248 220 269Previous analysis of Y08823 suggested that beta strands forming the DEBand non-twisted AGFCC′C″ beta sheets within the amino terminal domainwere predicted to be conserved between Y08823 and B7-1 (Ikemizu, S. etal. (2000) Immunity 12:51-60). Interestingly, the highest degree ofpredicted secondary structure conservation between the GL50 sequencesand Y08823 was also within the regions encompassing the DEB beta sheetsof the corresponding amino terminal domain. Predictions based on thesestructural homologies suggest that sequence identities in this regioncould provide key interdomain electrostatic contacts and conservehydropathicity within the interdomain core, resulting in a similarmolecular framework shared by the GL50 and B7 molecules (FIG. 16). Basedon these observations, chicken Y08823 was assessed for the ability tobind ICOS receptors. Sequences representing the mature Y08823 peptidewere obtained by RT-PCR and subcloned into an expression vector, whichupon transfection of COS cells, yielded a functional surface protein.Y08823 transfected cells were found to bind CTLA4-Ig but not tohICOS-mIgG2am nor mICOS-mIgG2am (FIG. 17). Although it cannot be ruledout that the binding of Y08823 to ICOS occurs at levels below detection,based on the assay conditions used here, it is not likely that theGL50-like protein Y08823 can cross-function as a ligand for human ormouse ICOS receptors.

Structural and genetic similarity suggests that B7/GL50 type proteinsare conserved across extreme phylogenetic boundaries, and implicit inthis interpretation is that mechanistic pathways utilizing theseproteins are also shared. The evidence that these proteins have similarfunctions in T cell signaling raise the question of the absolute numberand the origins of costimulatory ligands, their cognate receptors, andderivative spliced variants that exist. Other proteins that fit into theB7 Ig-superfamily structure include MOG and butyrophilin, but theseproteins have not been determined to participate as ligands in anycostimulatory pathway (Henry, J. et al. (1999) Immunol. Today 20:285-8).With the sequence availability of chromosome 21 (Hattori, M. et al.(2000) Nature 405:311-9), the genomic organization of the humanICOS-ligand was determined, indicating the presence of at least 2 splicevariants in the form of hGL50 (Ling, V. et al. (2000) J. Immunol.164:1653-7) and KIAA clone 0653 (Genbank Accession No. AB014453). Amongthe members of the B7-like genes, the genomic structure of B7-1, B7-2,butyrophilin, and hGL50 have been reported. Although the absolute numberof exons that comprise these genes varies from 5 to 12, these genesshare structure, in that distinct exons encode the two Ig-likeextracellular domains, one exon encodes the transmembrane domain, andmultiple exons encode the cytoplasmic domain (e.g., two exons for hGL50,two exons for B7-2 (Jellis, C. E. et al. (1995) Immunogenetics 42:85-9;Borriello, F. et al. (1995) J. Immunol. 155:5490-7), one to two exonsfor B7-1 (Borriello, F. et al. (1994) J. Immunol. 153:5038-48), andthree exons for butyrophilin (Ogg, S. L. et al. (1996) Mamm. Genome7:900-5)). For KIAA0653, the splice junction between exons encodingcytoplasmic domains 1 and 2 is not used, resulting in a read-through of2.9 kb into the putative intron 6. Upon alignment of KIAA0653 withchromosome 21 BAC clone HS21C098, the alternative 3′ cytoplasmic domainof KIAA0653 was not found to be in agreement: eight sequencediscrepancies were found, comprised of 7 mismatches and one 17 bpdeletion. In contrast, exon sequence alignment of human GL50 to HS21C098revealed no sequence dissimilarities up to and including thepolyadenylation site. The examples set forth above show that human GL50,mGL50-1, and variant mGL50-2 show some amino acid sequence identity nearthe splice site for cytoplasmic domains 1 and 2 (mGL50-1 residues316-318: E-L-T; FIG. 16). The shared point of splice variation betweenhGL50/AB014553 and between mGL50-1/mGL50-2 suggests the potential of aconserved mechanism that allows or promotes alternative splicing ofcytoplasmic domain 2, perhaps to offer alternate signaling through thecombinatorial addition of alternate functional domains. The observationthat mGL50-2 and the original mGL50-1 are transcribed with differingtissue specificity supports the notion that regulation of thesemolecules in cell signaling is dependent on physiological locale andactivation state.

The existence of a conserved intracellular motif between mammalian GL50and avian Y08823, along with the presence of multiple forms of GL50 withdivergent carboxyl regions, further suggests that differences in theintracellular domain of these molecules may lead to distinct signalingfunctions. This is further supported by the presence of 3 additionaltyrosine residues found in the intracellular domain of mGL50-2, inaddition to the 2 shared with mGL50-1. This contrasts with the structureof B7-1 and B7-2, where the intracellular regions lack any obviousconserved sequences and have been deleted without impairment ofcostimulatory activity, suggesting that intracellular signaling is not akey feature of these B7 proteins (Brunschwig, E. B. et al. (1995) J.Immunol. 155:5498-505). The conserved motif of hGL50, although predictedto be in the intracellular portion of the molecule by hydrophobicityanalysis, was found to be encoded by the exon 5 transmembrane domain,and not the exon 6 cytoplasmic domain-1. In the chicken Y08823 cDNAclone, sequence homology terminates within three amino acid residuesfollowing the corresponding exon 6/cytoplasmic domain-1. If the genomicorganization of hGL50 is maintained in Y08823, where the conserved motifis encoded by the intracellular portion of the exon-5 transmembranedomain, then it is possible that DNA segments orthologous to exon 6 andexon 7, encoding cytoplasmic domains 1 and 2 in hGL50, may be completelyabsent in chicken. In the structural studies of the B7 cytoplasmicdomain, it is argued that those sequences may be completely dispensable(Brunschwig, E. B. et al. (1995) J. Immunol. 155:5498-505). However, thefact that alternate cytoplasmic exons are used in B7-1 and GL50 suggeststhat the addition of alternate exon domains may have occurred during thetime when the novel B7-like proteins were generated. The B7-likebutyrophilin proteins are encoded by a number of splice variants, thepredominant form of which contains a cytoplasmic domain 3 encoding aintracellular Ring finger motif which is perhaps used in transducingsignaling from this molecule (Ogg, S. L. et al. (1996) Mamm. Genome7:900-5). These observations support the idea that other ligand typemolecules, such as GL50 and Y08823, with the conserved intracellularmotif from exon-5 and other cytoplasmic domains, may have alternateroles as signal delivery and a signal receptor molecules, depending onthe environmental millieu in which the is cell is found.

To clearly define the cell subsets that show surface expression of GL50,comparative phenotyping of RAG1 −/− and Balb/C splenocyte subsets wasperformed. The examples set forth above show that freshly isolated CD4+and CD8+ cells, as well as RAG1 −/− CD11c+ cells containedsubpopulations of ICOS-ligand expressing cells. These results aredistinct from previous studies where ICOS-ligand was reported to beabsent in T-cell lines (Aicher, A. et al. (2000) J. Immunol.164(9):4689-96) and some dendritic cell lines (Yoshinaga, S. K. et al.(1999) Nature 402:827-32). RT-PCR analysis of purified cell subsetsconfirmed that both GL50-1 and GL50-2 were expressed in the same cellssuggesting that both transcripts may contribute to the surface displayof ICOS binding. In addition to antigen presenting cells, it wasdemonstrated that the initial expression of costimulatory ligands occursearly in the ES cell model of embryonic development with the presence ofB7-1 and GL50-1 transcripts in undifferentiated cells and in embryoidbodies cultured 10 days in vitro Ling, V. et al. (1998) Exp. Cell Res.241:55-65). In this study, it is further demonstrated that by RNAanalysis, GL50-2 transcripts are found within these tissues. By day 9 ofembryoid body differentiation, emergent hematopoietic cellsphenotypically resemble yolk sac hematopoietic progenitors in vivo, asevidenced by the potential of c-kit+/PECAM-1+ cells to produce mixedhematopoietic progenitors and CD45+ cells to produce macrophageprogenitors in colony-forming assays (Ling, V. and Neben, S. (1997) J.Cell Physiol. 171:104-15; Ling, V. et al. (1997) Eur. J. Immunol.27:509-14). These CD45+ cells were also found to be B7-1+ and B7-2+,strongly suggesting costimulatory ligand expression occurs very early inlymphopoiesis. Correspondingly, high levels of GL50-1 and GL50-2expression were found in sites of embryonic hematopoiesis such asembryonic day yolk sac and fetal liver. It is noteworthy thatICOS-ligand is inducible in embryonic fibroblast cultures, a cell typederived from a time point prior to definitive lymphopoiesis, suggestingthat the mechanism for costimulatory signaling cascade may be poisedindependently of the initial formation of adaptive immune response. Ithas been postulated that metazoans share common evolvable pathways thatoccur at the phylotypic stage of embyrogenesis, and that certain corephysiological processes which have special properties relevant tocomplex development are reflected during this time period of embryonicdevelopment and later in adult physiology (Kirschner, M. and Gerhart, J.(1998) Proc. Natl. Acad. Sci. USA 95:8420-7). It remains to bedetermined whether costimulatory ligands are part of some core processesutilized by both embryos and adult systems.

Despite the large genetic distance between the B7 family members, thefact that primate and rodent B7-1 and B7-2 retain cross-binding to CTLA4and CD28 across phylogenetic lines suggests tolerance of nucleotidereplacement within these signaling molecules through the time course ofnatural history. To compare the phylogenetic divergence pattern betweencostimulatory ligands and their receptors, protein sequences of CTLA4(Genbank Accession Nos. NM_(—)009843 and NM_(—)005214), CD28 (AccessionNos. NM_(—)007642, NM_(—)006139, and X67915), and ICOS (GenbankAccession No. AJ250559 and Genseq Accession No. V53199) receptors frommouse, human and chicken were analyzed. When represented in graphicalformat, the genetic distance values of these receptors (Table 6)revealed a pattern (FIG. 21) in which distances between ICOS and CD28proteins were closer than distances between ICOS and CTLA4. TABLE 6Alignment Scores between ICOS, CTLA4 and CD28 Percent Sequence IdentityhICOS mICOS hCTLA4 mCTLA4 hCD28 mCD28 chCD28 Genetic hICOS — 69 21 20 2824 21 Distance mICOS 41 — 17 16 25 21 20 hCTLA4 250 368 — 74 30 29 32mCTLA4 272 466 33 — 31 32 31 hCD28 175 205 165 154 — 67 50 mCD28 217 257167 149 44 — 48 chCD28 246 278 152 156 79 85 —

When comparing receptor sequence relationships between species, distancevalues for human CD28/ICOS (176) were smaller than those for mouseCD28/ICOS (257). Likewise, human CTLA4/ICOS distance values (261) werealso found to be smaller than mouse CTLA4/ICOS distances (405). Thesedata suggest that structure of ICOS molecule is more likely derived fromthe form of CD28 rather than CTLA4. In contrast, phylogenetic analysisof the costimulatory ligands demonstrated that the distance valuesbetween GL50 and B7-1 (243-282) were nearly equivalent to the distancevalues between GL50 and B7-2 (200-270). Y08823 was found to exhibithigher sequence identity and lower genetic distance (36-37%; 131-138) tomouse and human GL50 proteins than to B7 proteins (23-30%, 230-310). Thenear-equivalent genetic distances between the GL50 and the B7-1/B7-2family members and the non-equivalent genetic distance between the ICOSand the CD28/CTLA4 family members implies that theevolutionary/functional constraints guiding the receptor family isdifferent from those guiding the ligand family.

Phylogenetic sequence relationships may reflect genomic placement ofthese molecules: B7-1 and B7-2 co-localize to mouse chromosome 16 andhuman chromosome 3, while CTLA4, CD28, and ICOS co-localize to mousechromosome 1 and to human chromosome 2q33. In contrast, the GL50 geneticloci are not linked to the B7 loci; human GL50 is located at chromosome21q22 (Hattori, M. et al. (2000) Nature 405:311-9) while mouse GL50 islocated on chromosome 10. By TFastX analysis, no additional GL50-likehomologs were identified in chromosome 21, suggesting that GL50 may notexist as a family of genes clustered together like B7-1 and B7-2. Withrespect to Y08823, it is not clear whether this molecule is a trueortholog of B7-1 or whether Y08823 represents a novel B7-like moleculewhose ortholog has not been defined in mammalian systems. However, fromthe 23-30% sequence identity shared between B7s and Y08823, includingmultiple amino acid replacements at charged residue sites, it wassurprising that these proteins retain functional crossbinding to CTLA4(O'Regan, M. N. et al. (1999) Immunogenetics 49:68-71). The unexpectedresult of Y08823 bearing stronger structural resemblance to GL50, yetretaining binding properties characteristic of B7-1 and B7-2, suggeststhat structural and functional constraints to the divergence of thesecostimulatory ligands are low.

Numerous scenarios may account for the differing genetic distancesmeasured between receptor families and ligand families. It is possiblethat the genes encoding the GL50/B7 family of proteins emerged earlierthan genes encoding the CD28/CTLA4 receptors. The formation of genesencoding the ICOS receptor may have arisen later during phylogeny andmay be based on the structure of CD28, thus resulting in a greatersimilarity to CD28 than CTLA4 molecules. This hypothesis may account forthe numerous B7-like proteins that exist, while relatively few CD28-likereceptors have been described. It is notable that certain exons of CTLA4retain remarkable sequence constraint, even at the level of synonymousDNA mutations, suggesting the presence of a yet-to-be-defined mechanismthat protects that locus from random mutations (Ling, V. et al. (1999)Genomics 60:341-355). It may be that a mutation constraining mechanismregulates the costimulatory receptor region over the length of theCTLA4/CD28/ICOS loci, or that the added selection pressure upon theintracellular signaling domain of these receptors is sufficient tomaintain a lower rate of divergence.

Costimulatory ligands and receptors belong to the Ig-superfamily ofproteins, which have been defined as those proteins that share homologyto immunoglobulins at the 10-20% range, with characteristic intrachaindisulfide bonds. Ig-superfamily proteins are widely distributed amongproteins of different functions and between vertebrate phylogenies. Theappearance of arthropods and chordates dates back 600 million years, andit has been suggested that molecules representing the putativeprogenitors of the Ig-superfamily are even more ancient, probably beingpresent in the acoelomates such as flatworms and nematodes. The notionthat the Ig-superfamily of proteins is at least as ancient is supportedby the finding that some Ig-like proteins such as N-CAM are found inmammals as well as insects. The immunological “big bang” event(Marchalonis, J. J. et al. (1998) Immunol. Rev. 166:103-22, andreferences therein) which gave rise to the Ig-based, combinatorialadaptive immune system theoretically appeared during the emergence ofjawed fish 450 million years ago over a geologically brief time span of10-20 million years. Currently, no mechanism by which the immunoglobulinsystem may have emerged from the Ig-superfamily of molecules has beenclearly defined. However, theories have been proposed that suggest thatgenes encoding Ig-domains and recombinase enzymes necessary for thecombinatorial immune system were horizontally transferred on asufficiently large enough scale to offer a selective advantage(Bernstein, R. M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:9454-9).Notably absent is a foundation for a comprehensive biochemical frameworkincorporating the salient signaling features of Ig-superfamilycostimulatory molecules which serve to trigger cell activation, promoteimmunoglobulin molecule maturation, and influence class switching.Although it is not currently known whether extant members of the ancientchondricthyes lineage such as sharks have costimulatory molecules, thefact that costimulation related proteins such as CD28 and Y08823 arepresent in chickens suggests that some type of costimulatory pathway waspresent in members of the avian lineage, which emerged at least 300million years ago (Burt, D. W. et al. (1999) Nature 402:411-3), openingthe possibility that the Y08823 molecule represents a contemporarycousin to both the GL50 and B7 molecules with a stronger resemblance toa prototypic costimulatory ligand, rather than being a true ortholog ofeither GL50 or B7. In contrast to the avian lineage, it is postulatedthat the mouse and human lineages separated approximately 100 millionyears ago, with the mouse genome undergoing extensive chromosomalrearrangements (Burt, D. W. et al. (1999) Nature 402:411-3) compared tothose seen in chickens and humans. It is not known whether theserearrangements may have led to the chromosomal separation between the B7family members and the genes encoding GL50 molecules. It is also notknown if avian ICOS or variants thereof exist.

Example 12 Soluble GL50 can Costimulate Human T Cells

The ability of soluble hGL50-mIgG2am to costimulate human T cells wasdetermined using a T cell costimulation assay. Naïve CD4+ T cells werepurified and plated at 10% cells per well. Cells were stimulated withanti-CD3 on beads, using one bead per cell and 1 or 2 μg anti-CD3 per10⁷ beads. Cells were treated with hGL50-mIgG2am on beads, using onebead per cell and 3 μg hGL50-mIgG2am per 10⁷ beads. CD28 signaling wasprovided (using anti-CD28 (Pharmingen)) or stimulated to determinewhether modulation of CD28 mediated costimulation had any effect onhGL50-mIgG2am mediated costimulation.

IL-2 production, IL-10 production, and proliferation (³H incorporation)were assayed as indicators of costimulation. Cytokines and proliferationwere measured 72 hours after stimulation.

As shown in FIG. 22, hGL50-mIgG2am (also called hGL50.Fc) cancostimulate T cells, as shown by the increase in proliferation as wellas the induction of IL-2 and IL-10 production. In the presence ofantibodies to CD28, which induces CD28 mediated costimulation, IL-2production is also induced. FIG. 23 shows the effects of varyingconcentrations of anti-CD3 and anti-CD28 on proliferation and cytokineproduction.

FIG. 24 shows that adding anti-CD28 to T cells stimulated with anti-CD3or anti-CD3 and soluble hGL50-mIgG2am (to stimulate CD28 mediatedcostimulation) induces IL-2 production, but does not influence hGL50mediated IL-10 production.

Example 13 Treatment of Murine Tumors Using ICOS/GL50 PathwayStimulation

As of yet, the role of ICOS/GL50 costimulation in the generation ofantitumor responses has not been reported. In this study, the relativeefficacy of ICOS/GL50 costimulation was compared to CD28/B7costimulation in various murine tumor models. For systemic treatment oftumor bearing animals, murine B7.2-IgG2a and GL50-IgG2a fusion proteinswere generated, which consist of the extracellular domain of B7.2 orGL50, respectively, and the Fc portion of murine IgG2a. Murine isotypeIgG2a was used as a control. Mice bearing MethA or B16F1 melanoma tumorswere treated subcutaneously with 50 μg/injection of GL50-IgG2a orB7.2-IgG2a fusion protein twice weekly for three weeks. In the MethAmodel, treatment with B7.2-IgG2a resulted in up to 100% tumor regression(FIG. 25A) and cure of the mice (FIG. 25E), and treatment withGL50-IgG2a resulted in up to 60-90% cure of mice (FIG. 25E) and in 40%significant tumor growth delay (FIG. 25D). In the B16F1 melanoma,systemic treatment with either protein led to comparable significanttumor growth delay. In both tumor models, control IgG2a treatment had noeffect (FIGS. 25A, C, and E). In tumor vaccines studies, the B16F1melanoma and the MB49 bladder carcinoma models were used; Tumor cellswere transduced with a vector containing the EF-1 alpha promoterexpressing either murine B7.1 or GL50, and G418 (neomycin) selectedtumor cells were injected subcutaneously for in vivo tumorigenicityexperiments. Expression of GL50 and B7-1 on tumor cells was determinedby FACS analysis using an anti-mB7-1 monoclonal antibody (Pharmingen,clone 16-10A1) or ICOS-IgG2a fusion protein. The results demonstrate:(i) in the B16F1 model, 40% of the mice injected with GL50 expressingtumor cells and 20% of the mice injected with B7.1 expressing tumorcells reject their tumors (FIG. 31A); (ii) in the MB49 model, 30% of themice injected with GL50 expressing tumor cells and 10% of the miceinjected with B7.1 expressing tumor cells reject their tumors (FIG.31B). These results indicate that enhanced in vivo ICOS/GL50interactions, provided either by soluble GL50-IgG or GL50 expression ontumor cells, has significant antitumor activity that is comparable tothe well described antitumor efficacy of the CD28/B7 pathway in murinetumor models.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-18. (canceled)
 19. An antibody which selectively binds to an isolatedpolypeptide selected from the group consisting of: a) an isolatedfragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 4, or 6, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:2, 4, or 6; b) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 4, or 6, wherein the polypeptide is encoded by a nucleic acidmolecule which hybridizes to a nucleic acid molecule consisting of SEQID NO:1, 3, or 5 under stringent conditions; c) a polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 50% identical to a nucleic acid molecule comprisingthe coding region of the nucleotide sequence of SEQ ID NO:1, 3, or 5; d)a polypeptide comprising an amino acid sequence which is at least 50%identical to the amino acid sequence of SEQ ID NO:2, 4, or
 6. 20-34.(canceled)